U.S. patent application number 12/302196 was filed with the patent office on 2010-07-08 for serum/plasma micronas and uses thereof.
Invention is credited to Yi Ba, Jiangning Chen, Xi Chen, Jin Wang, Ke Zeng, Chenyu Zhang, Hongjie Zhang, Junfeng Zhang.
Application Number | 20100173288 12/302196 |
Document ID | / |
Family ID | 40590521 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100173288 |
Kind Code |
A1 |
Zhang; Chenyu ; et
al. |
July 8, 2010 |
SERUM/PLASMA MICRONAS AND USES THEREOF
Abstract
This invention provides a combination of microRNAs for
evaluating the physiological and/or pathological condition of a
subject, wherein the combination comprises all detectable microRNAs
stably existing in the serum/plasma of a subject; and a method for
evaluating the physiological and/or pathological condition of a
subject, wherein the method includes determining all detectable
microRNAs stably existing in the serum/plasma of a subject; and a
kit for evaluating the physiological and/or pathological condition
of a subject, wherein the kit contains the tools for determining
all detectable microRNAs that stably existing in the serum/plasma
of a subject; and a biochip for evaluating the physiological and/or
pathological condition of a subject, wherein the biochip contains
the components for determining all detectable microRNAs stably
existing in the serum/plasma of a subject. The aforementioned
combination, method, kit and biochip can be used for diagnosis as
well as differentially diagnosis of diseases including various
tumors; various acute/chronic infectious diseases, e.g. viral
diseases such as viral influenza, viral hepatitis, AIDS, SARS,
bacterial diseases such as tuberculosis, bacterial pneumonia, and
other acute/chronic infectious diseases caused by various
pathogenic microorganisms; other acute/chronic diseases such as
diseases of respiratory system, diseases of immune system, diseases
of blood and hematopoietic system, diseases of circulatory system
such as cardio-cerebrovascular diseases, metabolic diseases of
endocrine system, diseases of digestive system, diseases of nervous
system, diseases of urinary system, diseases of reproductive system
and diseases of locomotor system, prediction of complications
occurrence and malignant diseases relapse, evaluation of
therapeutic effects, screening of pharmaceutical active
ingredients, assessment of drug efficacy as well as forensic
authentication and prohibited drug inspection and the like,
possessing a number of advantages such as extensive detection
spectrum, high sensitivity, low cost, convenience for sampling,
ease for sample preservation, etc. The said method can be widely
used in work related to general survey of diseases and so on,
improve the low-specificity and low-sensitivity caused by
individual differences which single markers are difficult to
overcome, significantly increasing the clinical detection rate of
diseases, all of which make it become an effective means for
diagnosing diseases in an early phase.
Inventors: |
Zhang; Chenyu; (Jiangsu,
CN) ; Zhang; Junfeng; (Jiangsu, CN) ; Chen;
Xi; (Jiangsu, CN) ; Ba; Yi; (Tianjin, CN)
; Chen; Jiangning; (Jiangsu, CN) ; Wang; Jin;
(Jiangsu, CN) ; Zeng; Ke; (Jiangsu, CN) ;
Zhang; Hongjie; (Jiangsu, CN) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Family ID: |
40590521 |
Appl. No.: |
12/302196 |
Filed: |
December 6, 2007 |
PCT Filed: |
December 6, 2007 |
PCT NO: |
PCT/CN07/03463 |
371 Date: |
November 24, 2008 |
Current U.S.
Class: |
435/6.1 ;
435/287.2 |
Current CPC
Class: |
C12Q 1/6809 20130101;
C12Q 2600/158 20130101; C12Q 1/6886 20130101; C12Q 2600/178
20130101; C12Q 1/6883 20130101; C12Q 1/6809 20130101; C12Q 1/6876
20130101; C12Q 2525/207 20130101 |
Class at
Publication: |
435/6 ;
435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 2, 2007 |
CN |
200710134620.4 |
Claims
1. A combination of microRNAs for evaluating the physiological
and/or pathological condition of a subject, wherein the combination
comprises all detectable microRNAs stably existing in the
serum/plasma of the subject.
2. The combination according to claim 1, characterized in that said
all detectable microRNAs stably existing in the serum/plasma of a
subject are all mature microRNAs in human serum/plasma.
3. The combination according to claim 1, characterized in that said
all detectable microRNAs stably existing in the serum/plasma of a
subject are let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g,
let-7i, miR-1, miR-100, miR-101, miR-103, miR-105, miR-106a,
miR-106b, miR-107, miR-10a, miR-10b, miR-122a, miR-124a, miR-125a,
miR-125b, miR-126, miR-126*, miR-127, miR-128a, miR-128b, miR-129,
miR-130a, miR-130b, miR-132, miR-133a, miR-133b, miR-134, miR-135a,
miR-135b, miR-136, miR-137, miR-138, miR-139, miR-140, miR-141,
miR-142-3p, miR-142-5p, miR-143, miR-144, miR-145, miR-146a,
miR-146b, miR-147, miR-148a, miR-148b, miR-149, miR-150, miR-151,
miR-152, miR-153, miR-154, miR-154*, miR-155, miR-15a, miR-15b,
miR-16, miR-17-3p, miR-17-5p, miR-181a, miR-181b, miR-181c,
miR-181d, miR-182, miR-182*, miR-183, miR-184, miR-185, miR-186,
miR-187, miR-188, miR-189, miR-18a, miR-18a*, miR-18b, miR-190,
miR-191, miR-191*, miR-192, miR-193a, miR-193b, miR-194, miR-195,
miR-196a, miR-196b, miR-197, miR-198, miR-199a, miR-199a*,
miR-199b, miR-19a, miR-19b, miR-200a, miR-200a*, miR-200b,
miR-200c, miR-202, miR-202*, miR-203, miR-204, miR-205, miR-206,
miR-208, miR-20a, miR-20b, miR-21, miR-210, miR-211, miR-212,
miR-213, miR-214, miR-215, miR-216, miR-217, miR-218, miR-219,
miR-22, miR-220, miR-221, miR-222, miR-223, miR-224, miR-23a,
miR-23b, miR-24, miR-25, miR-26a, miR-26b, miR-27a, miR-27b,
miR-28, miR-296, miR-299-3p, miR-299-5p, miR-29a, miR-29b, miR-29c,
miR-301, miR-302a, miR-302a*, miR-302b, miR-302b*, miR-302c,
miR-302c*, miR-302d, miR-30a-3p, miR-30a-5p, miR-30b, miR-30c,
miR-30d, miR-30e-3p, miR-30e-5p, miR-31, miR-32, miR-320, miR-323,
miR-324-3p, miR-324-5p, miR-325, miR-326, miR-328, miR-329, miR-33,
miR-330, miR-331, miR-335, miR-337, miR-338, miR-339, miR-33b,
miR-340, miR-342, miR-345, miR-346, miR-34a, miR-34b, miR-34c,
miR-361, miR-362, miR-363, miR-363*, miR-365, miR-367, miR-368,
miR-369-3p, miR-369-5p, miR-370, miR-371, miR-372, miR-373,
miR-373*, miR-374, miR-375, miR-376a, miR-376a*, miR-376b, miR-377,
miR-378, miR-379, miR-380-3p, miR-380-5p, miR-381, miR-382,
miR-383, miR-384, miR-409-3p, miR-409-5p, miR-410, miR-411,
miR-412, miR-421, miR-422a, miR-422b, miR-423, miR-424, miR-425,
miR-425-5p, miR-429, miR-431, miR-432, miR-432*, miR-433, miR-448,
miR-449, miR-450, miR-451, miR-452, miR-452*, miR-453, miR-455,
miR-483, miR-484, miR-485-3p, miR-485-5p, miR-486, miR-487a,
miR-487b, miR-488, miR-489, miR-490, miR-491, miR-492, miR-493,
miR-493-3p, miR-494, miR-495, miR-496, miR-497, miR-498, miR-499,
miR-500, miR-501, miR-502, miR-503, miR-504, miR-505, miR-506,
miR-507, miR-508, miR-509, miR-510, miR-511, miR-512-3p,
miR-512-5p, miR-513, miR-514, miR-515-3p, miR-515-5p, miR-516-3p,
miR-516-5p, miR-517*, miR-517a, miR-517b, miR-517c, miR-518a,
miR-518a-2*, miR-518b, miR-518c, miR-518c*, miR-518d, miR-518e,
miR-518f, miR-518f*, miR-519a, miR-519b, miR-519c, miR-519d,
miR-519e, miR-519e*, miR-520a, miR-520a*, miR-520b, miR-520c,
miR-520d, miR-520d*, miR-520e, miR-520f, miR-520g, miR-520h,
miR-521, miR-522, miR-523, miR-524, miR-524*, miR-525, miR-525*,
miR-526a, miR-526b, miR-526b*, miR-526c, miR-527, miR-532,
miR-542-3p, miR-542-5p, miR-544, miR-545, miR-548a, miR-548b,
miR-548c, miR-548d, miR-549, miR-550, miR-551a, miR-552, miR-553,
miR-554, miR-555, miR-556, miR-557, miR-558, miR-559, miR-560,
miR-561, miR-562, miR-563, miR-564, miR-565, miR-566, miR-567,
miR-568, miR-569, miR-570, miR-571, miR-572, miR-573, miR-574,
miR-575, miR-576, miR-577, miR-578, miR-579, miR-580, miR-581,
miR-582, miR-583, miR-584, miR-585, miR-586, miR-587, miR-588,
miR-589, miR-590, miR-591, miR-592, miR-593, miR-594, miR-595,
miR-596, miR-597, miR-598, miR-599, miR-600, miR-601, miR-602,
miR-603, miR-604, miR-605, miR-606, miR-607, miR-608, miR-609,
miR-610, miR-611, miR-612, miR-613, miR-615, miR-616, miR-617,
miR-618, miR-619, miR-620, miR-621, miR-622, miR-623, miR-624,
miR-625, miR-626, miR-627, miR-628, miR-629, miR-630, miR-631,
miR-632, miR-633, miR-634, miR-635, miR-636, miR-637, miR-638,
miR-639, miR-640, miR-641, miR-642, miR-643, miR-644, miR-645,
miR-646, miR-647, miR-648, miR-649, miR-650, miR-651, miR-652,
miR-653, miR-654, miR-655, miR-656, miR-657, miR-658, miR-659,
miR-660, miR-661, miR-662, miR-663, miR-7, miR-9, miR-9*, miR-92,
miR-93, miR-95, miR-96, miR-98, miR-99a and miR-99b.
4. The combination according to claim 1, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to determine physiological and/or pathological condition
of the subject after being administrated a test sample.
5. The combination according to claim 4, characterized in that the
combination is useful for screening the test sample for the
activities on the prevention and/or treatment of diseases.
6. The combination according claim 1, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to diagnose and/or differentially diagnose the diseases
of the subject.
7. The combination according to claim 1, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to evaluate the effectiveness of treating the diseases
of the subject.
8. The combination of according to claim 1, characterized in that
said evaluating the physiological and/or pathological condition of
a subject is to predict the occurrence of diseases of the
subject.
9. The combination according to claim 8, characterized in that the
occurrence of diseases is the occurrence of complications and/or
the relapse of malignant diseases.
10. The combination according to claim 5, characterized in that the
diseases include various tumors; various acute and chronic
infectious diseases, e.g. viral diseases such as viral influenza,
viral hepatitis, AIDS, SARS, bacterial diseases such as
tuberculosis, bacterial pneumonia, and other acute and chronic
infectious diseases caused by various pathogenic microorganisms;
other acute and chronic diseases such as diseases of respiratory
system, diseases of immune system, diseases of blood and
hematopoietic system, diseases of circulatory system such as
cardio-cerebrovascular diseases, metabolic diseases of endocrine
system, diseases of digestive system, diseases of nervous system,
diseases of urinary system, diseases of reproductive system and
diseases of locomotor system.
11. The combination according to claim 1, characterized in that the
combination is useful for detecting the subject for prohibited
drugs-taking.
12. The combination according to claim 1, characterized in that the
serum/plasma derives from living body, tissues, organs and/or
corpuses of the subject.
13. A method for evaluating the physiological and/or pathological
condition of a subject, wherein the method comprises determining
all detectable microRNAs stably existing in serum/plasma of the
subject.
14. The method according to claim 13, characterized in that said
all detectable microRNAs stably existing in serum/plasma of a
subject are all mature microRNAs in human serum/plasma.
15. The method according to claim 13, characterized in that said
all detectable microRNAs stably existing in serum/plasma of a
subjects are let-7a, let-7b, let-7c, let-7d, let-7e, let-7f,
let-7g, let-7i, miR-1, miR-100, miR-101, miR-103, miR-105,
miR-106a, miR-106b, miR-107, miR-10a, miR-10b, miR-122a, miR-124a,
miR-125a, miR-125b, miR-126, miR-126*, miR-127, miR-128a, miR-128b,
miR-129, miR-130a, miR-130b, miR-132, miR-133a, miR-133b, miR-134,
miR-135a, miR-135b, miR-136, miR-137, miR-138, miR-139, miR-140,
miR-141, miR-142-3p, miR-142-5p, miR-143, miR-144, miR-145,
miR-146a, miR-146b, miR-147, miR-148a, miR-148b, miR-149, miR-150,
miR-151, miR-152, miR-153, miR-154, miR-154*, miR-155, miR-15a,
miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-181a, miR-181b,
miR-181c, miR-181d, miR-182, miR-182*, miR-183, miR-184, miR-185,
miR-186, miR-187, miR-188, miR-189, miR-18a, miR-18a*, miR-18b,
miR-190, miR-191, miR-191*, miR-192, miR-193a, miR-193b, miR-194,
miR-195, miR-196a, miR-196b, miR-197, miR-198, miR-199a, miR-199a*,
miR-199b, miR-19a, miR-19b, miR-200a, miR-200a*, miR-200b,
miR-200c, miR-202, miR-202*, miR-203, miR-204, miR-205, miR-206,
miR-208, miR-20a, miR-20b, miR-21, miR-210, miR-211, miR-212,
miR-213, miR-214, miR-215, miR-216, miR-217, miR-218, miR-219,
miR-22, miR-220, miR-221, miR-222, miR-223, miR-224, miR-23a,
miR-23b, miR-24, miR-25, miR-26a, miR-26b, miR-27a, miR-27b,
miR-28, miR-296, miR-299-3p, miR-299-5p, miR-29a, miR-29b, miR-29c,
miR-301, miR-302a, miR-302a*, miR-302b, miR-302b*, miR-302c,
miR-302c*, miR-302d, miR-30a-3p, miR-30a-5p, miR-30b, miR-30c,
miR-30d, miR-30e-3p, miR-30e-5p, miR-31, miR-32, miR-320, miR-323,
miR-324-3p, miR-324-5p, miR-325, miR-326, miR-328, miR-329, miR-33,
miR-330, miR-331, miR-335, miR-337, miR-338, miR-339, miR-33b,
miR-340, miR-342, miR-345, miR-346, miR-34a, miR-34b, miR-34c,
miR-361, miR-362, miR-363, miR-363*, miR-365, miR-367, miR-368,
miR-369-3p, miR-369-5p, miR-370, miR-371, miR-372, miR-373,
miR-373*, miR-374, miR-375, miR-376a, miR-376a*, miR-376b, miR-377,
miR-378, miR-379, miR-380-3p, miR-380-5p, miR-381, miR-382,
miR-383, miR-384, miR-409-3p, miR-409-5p, miR-410, miR-411,
miR-412, miR-421, miR-422a, miR-422b, miR-423, miR-424, miR-425,
miR-425-5p, miR-429, miR-431, miR-432, miR-432*, miR-433, miR-448,
miR-449, miR-450, miR-451, miR-452, miR-452*, miR-453, miR-455,
miR-483, miR-484, miR-485-3p, miR-485-5p, miR-486, miR-487a,
miR-487b, miR-488, miR-489, miR-490, miR-491, miR-492, miR-493,
miR-493-3p, miR-494, miR-495, miR-496, miR-497, miR-498, miR-499,
miR-500, miR-501, miR-502, miR-503, miR-504, miR-505, miR-506,
miR-507, miR-508, miR-509, miR-510, miR-511, miR-512-3p,
miR-512-5p, miR-513, miR-514, miR-515-3p, miR-515-5p, miR-516-3p,
miR-516-5p, miR-517*, miR-517a, miR-517b, miR-517c, miR-518a,
miR-518a-2*, miR-518b, miR-518c, miR-518c*, miR-518d, miR-518e,
miR-518f, miR-518f*, miR-519a, miR-519b, miR-519c, miR-519d,
miR-519e, miR-519e*, miR-520a, miR-520a*, miR-520b, miR-520c,
miR-520d, miR-520d*, miR-520e, miR-520f, miR-520g, miR-520h,
miR-521, miR-522, miR-523, miR-524, miR-524*, miR-525, miR-525*,
miR-526a, miR-526b, miR-526b*, miR-526c, miR-527, miR-532,
miR-542-3p, miR-542-5p, miR-544, miR-545, miR-548a, miR-548b,
miR-548c, miR-548d, miR-549, miR-550, miR-551a, miR-552, miR-553,
miR-554, miR-555, miR-556, miR-557, miR-558, miR-559, miR-560,
miR-561, miR-562, miR-563, miR-564, miR-565, miR-566, miR-567,
miR-568, miR-569, miR-570, miR-571, miR-572, miR-573, miR-574,
miR-575, miR-576, miR-577, miR-578, miR-579, miR-580, miR-581,
miR-582, miR-583, miR-584, miR-585, miR-586, miR-587, miR-588,
miR-589, miR-590, miR-591, miR-592, miR-593, miR-594, miR-595,
miR-596, miR-597, miR-598, miR-599, miR-600, miR-601, miR-602,
miR-603, miR-604, miR-605, miR-606, miR-607, miR-608, miR-609,
miR-610, miR-611, miR-612, miR-613, miR-614, miR-615, miR-616,
miR-617, miR-618, miR-619, ma-620, miR-621, miR-622, miR-623,
miR-624, miR-625, miR-626, miR-627, miR-628, miR-629, miR-630,
miR-631, miR-632, miR-633, miR-634, miR-635, miR-636, miR-637,
miR-638, miR-639, miR-640, miR-641, miR-642, miR-643, miR-644,
miR-645, miR-646, miR-647, miR-648, miR-649, miR-650, miR-651,
miR-652, miR-653, miR-654, miR-655, miR-656, miR-657, miR-658,
miR-659, miR-660, miR-661, miR-662, miR-663, miR-7, miR-9, miR-9*,
miR-92, miR-93, miR-95, miR-96, miR-98, miR-99a and miR-99b.
16. The method according to claim 13, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to determine the physiological and/or pathological
condition of the subject after being administrated a test
sample.
17. The method according to claim 16, characterized in that the
method is useful for screening the test sample for the activities
on the prevention and/or treatment of diseases.
18. The method according to claim 13, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to diagnose and/or differentially diagnose the diseases
of the subject.
19. The method according to claim 13, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to evaluate the effectiveness of treating the diseases
of the subject.
20. The method according to claim 13, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to predict the occurrence of diseases of the
subject.
21. The method according to claim 20, characterized in that the
occurrence of diseases is the occurrence of complications and/or
the relapse of malignant diseases.
22. The method according to claim 17, characterized in that the
diseases include various tumors; various acute and chronic
infectious diseases e.g. viral diseases such as viral influenza,
viral hepatitis, AIDS, SARS; bacterial diseases such as
tuberculosis, bacterial pneumonia, and other acute and chronic
infectious diseases caused by various pathogenic microorganisms;
other acute and chronic diseases such as diseases of respiratory
system, diseases of immune system, diseases of blood and
hematopoietic system, diseases of circulatory system such as
cardio-cerebrovascular disease, metabolic diseases of endocrine
system, diseases of digestive system, diseases of nervous system,
diseases of urinary system, diseases of reproductive system and
diseases of locomotor system.
23. The method according to claim 13, characterized in that the
method is useful for detecting the subject for prohibited
drugs-taking.
24. The method according to claim 13, characterized in that the
method for determining all detectable microRNAs stably existing in
serum/plasma of a subject is one or more selected from the group
consisting of RT-PCR method, Real-time PCR method, Northern
blotting method, RNase protection assay, Solexa sequencing
technology and biochip method.
25. The method according to claim 24, characterized in that the
method is RT-PCR method which includes the following steps: (1)
extracting the total RNA from the serum/plasma of the subject, and
obtaining cDNA samples by RNA reverse transcription reaction; or
collecting serum/plasma samples from the subject and conducting
reverse transcription reaction with serum/plasma being a buffer so
as to prepare cDNA samples; (2) designing a primer by use of
microRNAs and conducting PCR reaction; (3) conducting agarose gel
electrophoresis of PCR products; and (4) observing agarose gel
under ultraviolet Lamp after EB staining.
26. The method according to claim 24, characterized in that the
method is Real-time PCR which includes the following steps: (1)
extracting the total RNA from the serum/plasma of the subject, and
obtaining cDNA samples by RNA reverse transcription reaction; or
collecting serum/plasma samples from the subject, preparing cDNA
samples by reverse transcription reaction with serum/plasma being a
buffer; (2) designing a primer by use of microRNAs; (3) adding a
fluorescent probe to conduct PCR reaction; (4) detecting and
comparing the variation in levels of microRNAs in the serum/plasma
samples relative to those of microRNAs in normal serum/plasma.
27. The method according to claim 13, characterized in that the
serum/plasma derives from living bodies, tissues, organs and/or
corpuses of the subject.
28. A kit for evaluating the physiological and/or pathological
condition of a subject, wherein the kit comprises the tools useful
for determining all detectable microRNAs stably existing in the
serum/plasma of the subject.
29. The kit according to claim 28, characterized in that the kit
comprises the primers of all mature microRNAs in human
serum/plasma.
30. The kit according to claim 28, characterized in that the kit
comprises the primers of let-7a, let-7b, let-7c, let-7d, let-7e,
let-7f, let-7g, let-7i, miR-1, miR-100, miR-101, miR-103, miR-105,
miR-106a, miR-106b, miR-107, miR-10a, miR-10b, miR-122a, miR-124a,
miR-125a, miR-125b, miR-126, miR-126*, miR-127, miR-128a, miR-128b,
miR-129, miR-130a, miR-130b, miR-132, miR-133a, miR-133b, miR-134,
miR-135a, miR-135b, miR-136, miR-137, miR-138, miR-139, miR-140,
miR-141, miR-142-3p, miR-142-5p, miR-143, miR-144, miR-145,
miR-146a, miR-146b, miR-147, miR-148a, miR-148b, miR-149, miR-150,
miR-151, miR-152, miR-153, miR-154, miR-154*, miR-155, miR-15a,
miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-181a, miR-181b,
miR-181c, miR-181d, miR-182, miR-182*, miR-183, miR-184, miR-185,
miR-186, miR-187, miR-188, miR-189, miR-18a, miR-18a*, miR-18b,
miR-190, miR-191, miR-191*, miR-192, miR-193a, miR-193b, miR-194,
miR-195, miR-196a, miR-196b, miR-197, miR-198, miR-199a, miR-199a*,
miR-199b, miR-19a, miR-19b, miR-200a, miR-200a*, miR-200b,
miR-200c, miR-202, miR-202*, miR-203, miR-204, miR-205, miR-206,
miR-208, miR-20a, miR-20b, miR-21, miR-210, miR-211, miR-212,
miR-213, miR-214, miR-215, miR-216, miR-217, miR-218, miR-219,
miR-22, miR-220, miR-221, miR-222, miR-223, miR-224, miR-23a,
miR-23b, miR-24, miR-25, miR-26a, miR-26b, miR-27a, miR-27b,
miR-28, miR-296, miR-299-3p, miR-299-5p, miR-29a, miR-29b, miR-29c,
miR-301, miR-302a, miR-302a*, miR-302b, miR-302b*, miR-302c,
miR-302c*, miR-302d, miR-30a-3p, miR-30a-5p, miR-30b, miR-30c,
miR-30d, miR-30e-3p, miR-30e-5p, miR-31, miR-32, miR-320, miR-323,
miR-324-3p, miR-324-5p, miR-325, miR-326, miR-328, miR-329, miR-33,
miR-330, miR-331, miR-335, miR-337, miR-338, miR-339, miR-33b,
miR-340, miR-342, miR-345, miR-346, miR-34a, miR-34b, miR-34c,
miR-361, miR-362, miR-363, miR-363*, miR-365, miR-367, miR-368,
miR-369-3p, miR-369-5p, miR-370, miR-371, miR-372, miR-373,
miR-373*, miR-374, miR-375, miR-376a, miR-376a*, miR-376b, miR-377,
miR-378, miR-379, miR-380-3p, miR-380-5p, miR-381, miR-382,
miR-383, miR-384, miR-409-3p, miR-409-5p, miR-410, miR-411,
miR-412, miR-421, miR-422a, miR-422b, miR-423, miR-424, miR-425,
miR-425-5p, miR-429, miR-431, miR-432, miR-432*, miR-433, miR-448,
miR-449, miR-450, miR-451, miR-452, miR-452*, miR-453, miR-455,
miR-483, miR-484, miR-485-3p, miR-485-5p, miR-486, miR-487a,
miR-487b, miR-488, miR-489, miR-490, miR-491, miR-492, miR-493,
miR-493-3p, miR-494, miR-495, miR-496, miR-497, miR-498, miR-499,
miR-500, miR-501, miR-502, miR-503, miR-504, miR-505, miR-506,
miR-507, miR-508, miR-509, miR-510, miR-511, miR-512-3p,
miR-512-5p, miR-513, miR-514, miR-515-3p, miR-515-5p, miR-516-3p,
miR-516-5p, miR-517*, miR-517a, miR-517b, miR-517c, miR-518a,
miR-518a-2*, miR-518b, miR-518c, miR-518c*, miR-518d, miR-518e,
miR-518f, miR-518f*, miR-519a, miR-519b, miR-519c, miR-519d,
miR-519e, miR-519e*, miR-520a, miR-520a*, miR-520b, miR-520c,
miR-520d, miR-520d*, miR-520e, miR-520f, miR-520g, miR-520h,
miR-521, miR-522, miR-523, miR-524, miR-524*, miR-525, miR-525*,
miR-526a, miR-526b, miR-526b*, miR-526c, miR-527, miR-532,
miR-542-3p, miR-542-5p, miR-544, miR-545, miR-548a, miR-548b,
miR-548c, miR-548d, miR-549, miR-550, miR-551a, miR-552, miR-553,
miR-554, miR-555, miR-556, miR-557, miR-558, miR-559, miR-560,
miR-561, miR-562, miR-563, miR-564, miR-565, miR-566, miR-567,
miR-568, miR-569, miR-570, miR-571, miR-572, miR-573, miR-574,
miR-575, miR-576, miR-577, miR-578, miR-579, miR-580, miR-581,
miR-582, miR-583, miR-584, miR-585, miR-586, miR-587, miR-588,
miR-589, miR-590, miR-591, miR-592, miR-593, miR-594, miR-595,
miR-596, miR-597, miR-598, miR-599, miR-600, miR-601, miR-602,
miR-603, miR-604, miR-605, miR-606, miR-607, miR-608, miR-609,
miR-610, miR-611, miR-612, miR-613, miR-614, miR-615, miR-616,
miR-617, miR-618, miR-619, miR-620, miR-621, miR-622, miR-623,
miR-624, miR-625, miR-626, miR-627, miR-628, miR-629, miR-630,
miR-631, miR-632, miR-633, miR-634, miR-635, miR-636, miR-637,
miR-638, miR-639, miR-640, miR-641, miR-642, miR-643, miR-644,
miR-645, miR-646, miR-647, miR-648, miR-649, miR-650, miR-651,
miR-652, miR-653, miR-654, miR-655, miR-656, miR-657, miR-658,
miR-659, miR-660, miR-661, miR-662, miR-663, miR-7, miR-9, miR-9*,
miR-92, miR-93, miR-95, miR-96, miR-98, miR-99a and miR-99b.
31. The kit according to claim 28, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to determine the physiological and/or pathological
condition of a subject after being administrated a test sample.
32. The kit according to claim 31, characterized in that the kit is
useful for screening the test sample for the activities on the
prevention and/or treatment of diseases.
33. The kit according to claim 28, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to diagnose and/or differentially diagnose the diseases
of the subject.
34. The kit according to claim 28, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to evaluate the effectiveness of treating the diseases
of the subject.
35. The kit according to claim 28, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to predict the occurrence of diseases of the
subject.
36. The kit according to claim 35, characterized in that the
occurrence of diseases is the occurrence of complications and/or
the relapse of malignant diseases.
37. The kit according to claim 32, characterized in that the
diseases include various tumors, various acute and chronic
infectious diseases e.g. viral diseases such as viral influenza,
viral hepatitis, AIDS, SARS, bacterial diseases such as
tuberculosis, bacterial pneumonia, and other acute and chronic
infectious diseases caused by various pathogenic microorganisms;
other acute and chronic diseases such as diseases of respiratory
system, diseases of immune system, diseases of blood and
hematopoietic system, diseases of circulatory system such as
cardio-cerebrovascular disease, metabolic diseases of endocrine
system, diseases of digestive system, diseases of nervous system,
diseases of urinary system, diseases of reproductive system and
diseases of locomotor system.
38. The kit according to claim 28, characterized in that the kit is
useful for detecting the subject for prohibited drugs-taking.
39. The kit according to claim 28, characterized in that the
serum/plasma of the subject are from living bodies, tissues, organs
and/or corpuses of the subject.
40. A biochip for evaluating the physiological and/or pathological
condition of a subject, wherein the biochip contains components
useful for determining all detectable microRNAs stably existing in
the serum/plasma of the subject.
41. The biochip according to claim 40, characterized in that the
biochip contains the probes for all mature microRNAs in human
serum/plasma.
42. The biochip according to claim 40, characterized in that the
biochip comprises the following probes: TABLE-US-00002 Corre-
sponding micro- Probes RNas Sequences of probes probe-let-7a let-7a
AACTATACAACCTACTACCTCA probe-let-7b let-7b AACCACACAACCTACTACCTCA
probe-let-7c let-7c AACCATACAACCTACTACCTCA probe-let-7d let-7d
ACTATGCAACCTACTACCTCT probe-let-7e let-7e ACTATACAACCTCCTACCTCA
probe-let-7f let-7f AACTATACAATCTACTACCTCA probe-let-7g let-7g
ACTGTACAAACTACTACCTCA probe-let-7i let-7i ACAGCACAAACTACTACCTCA
probe-miR-1 miR-1 TACATACTTCTTTACATTCCA probe-miR-100 miR-100
CACAAGTTCGGATCTACGGGTT probe-miR-101 miR-101 CTTCAGTTATCACAGTACTGTA
probe-miR-103 miR-103 TCATAGCCCTGTACAATGCTGCT probe-miR-105 miR-105
ACAGGAGTCTGAGCATTTGA probe-miR-106a miR-106a
GCTACCTGCACTGTAAGCACTTTT probe-miR-106b miR-106b
ATCTGCACTGTCAGCACTTTA probe-miR-107 miR-107 TGATAGCCCTGTACAATGCTGCT
probe-miR-10a miR-10a CACAAATTCGGATCTACAGGGTA probe-miR-10b miR-10b
ACAAATTCGGTTCTACAGGGTA probe-miR-122a miR-122a
ACAAACACCATTGTCACACTCCA probe-miR-124a miR-124a
TGGCATTCACCGCGTGCCTTAA probe-miR-125a miR-125a
CACAGGTTAAAGGGTCTCAGGGA probe-miR-125b miR-125b
TCACAAGTTAGGGTCTCAGGGA probe-miR-126 miR-126 GCATTATTACTCACGGTACGA
probe-miR-126* miR-126* CGCGTACCAAAAGTAATAATG probe-miR-127 miR-127
AGCCAAGCTCAGACGGATCCGA probe-miR-128a miR-128a
AAAAGAGACCGGTTCACTGTGA probe-miR-128b miR-128b
GAAAGAGACCGGTTCACTGTGA probe-miR-129 miR-129 GCAAGCCCAGACCGCAAAAAG
probe-miR-130a miR-130a ATGCCCTTTTAACATTGCACTG probe-miR-130b
miR-130b ATGCCCTTTCATCATTGCACTG probe-miR-132 miR-132
CGACCATGGCTGTAGACTGTTA probe-miR-133a miR-133a
ACAGCTGGTTGAAGGGGACCAA probe-miR-133b miR-133b
TAGCTGGTTGAAGGGGACCAA probe-miR-134 miR-134 CCCTCTGGTCAACCAGTCACA
probe-miR-135a miR-135a TCACATAGGAATAAAAAGCCATA probe-miR-135b
miR-135b CACATAGGAATGAAAAGCCATA probe-miR-136 miR-136
TCCATCATCAAAACAAATGGAGT probe-miR-137 miR-137
CTACGCGTATTCTTAAGCAATA probe-miR-138 miR-138 GATTCACAACACCAGCT
probe-miR-139 miR-139 AGACACGTGCACTGTAGA probe-miR-140 miR-140
CTACCATAGGGTAAAACCACT probe-miR-141 miR-141 CCATCTTTACCAGACAGTGTTA
probe-miR-142- miR-142- TCCATAAAGTAGGAAACACTACA 3p 3p
probe-miR-142- miR-142- GTAGTGCTTTCTACTTTATG 5p 5p probe-miR-143
miR-143 TGAGCTACAGTGCTTCATCTCA probe-miR-144 miR-144
CTAGTACATCATCTATACTGTA probe-miR-145 miR-145
AAGGGATTCCTGGGAAAACTGGAC probe-miR-146a miR-146a
AACCCATGGAATTCAGTTCTCA probe-miR-146b miR-146b
AGCCTATGGAATTCAGTTCTCA probe-miR-147 miR-147 GCAGAAGCATTTCCACACAC
probe-miR-148a miR-148a ACAAAGTTCTGTAGTGCACTGA probe-miR-148b
miR-148b ACAAAGTTCTGTGATGCACTGA probe-miR-149 miR-149
GGAGTGAAGACACGGAGCCAGA probe-miR-150 miR-150 CACTGGTACAAGGGTTGGGAGA
probe-miR-151 miR-151 CCTCAAGGAGCTTCAGTCTAGT probe-miR-152 miR-152
CCCAAGTTCTGTCATGCACTGA probe-miR-153 miR-153 TCACTTTTGTGACTATGCAA
probe-miR-154 miR-154 CGAAGGCAACACGGATAACCTA probe-miR-154*
miR-154* AATAGGTCAACCGTGTATGATT probe-miR-155 miR-155
CCCCTATCACGATTAGCATTAA probe-miR-15a miR-15a CACAAACCATTATGTGCTGCTA
probe-miR-15b miR-15b TGTAAACCATGATGTGCTGCTA probe-miR-16 miR-16
CGCCAATATTTACGTGCTGCTA probe-miR-17- miR-17- ACAAGTGCCTTCACTGCAGT
3p 3p probe-miR-17- miR-17- ACTACCTGCACTGTAAGCACTTTG 5p 5p
probe-miR-181a miR-181a ACTCACCGACAGCGTTGAATGTT probe-miR-181b
miR-181b CCCACCGACAGCAATGAATGTT probe-miR-181c miR-181c
ACTCACCGACAGGTTGAATGTT probe-miR-181d miR-181d
AACCCACCGACAACAATGAATGTT probe-miR-182 miR-182
TGTGAGTTCTACCATTGCCAAA probe-miR-182* miR-182*
TAGTTGGCAAGTCTAGAACCA probe-miR-183 miR-183 CAGTGAATTCTACCAGTGCCATA
probe-miR-184 miR-184 ACCCTTATCAGTTCTCCGTCCA probe-miR-185 miR-185
GAACTGCCTTTCTCTCCA probe-miR-186 miR-186 AAGCCCAAAAGGAGAATTCTTTG
probe-miR-187 miR-187 CGGCTGCAACACAAGACACGA probe-miR-188 miR-188
ACCCTCCACCATGCAAGGGATG probe-miR-189 miR-189
ACTGATATCAGCTCAGTAGGCAC probe-miR-18a miR-18a
TATCTGCACTAGATGCACCTTA probe-miR-18a* miR-18a*
AGAAGGAGCACTTAGGGCAGT probe-miR-18b miR-18b TAACTGCACTAGATGCACCTTA
probe-miR-190 miR-190 ACCTAATATATCAAACATATCA probe-miR-191 miR-191
AGCTGCTTTTGGGATTCCGTTG probe-miR-191* miR-191*
GGGGACGAAATCCAAGCGCAGC probe-miR-192 miR-192 GGCTGTCAATTCATAGGTCAG
probe-miR-193a miR-193a CTGGGACTTTGTAGGCCAGTT probe-miR-193b
miR-193b AAAGCGGGACTTTGAGGGCCAGTT probe-miR-194 miR-194
TCCACATGGAGTTGCTGTTACA probe-miR-195 miR-195 GCCAATATTTCTGTGCTGCTA
probe-miR-196a miR-196a CCAACAACATGAAACTACCTA probe-miR-196b
miR-196b CCAACAACAGGAAACTACCTA probe-miR-197 miR-197
GCTGGGTGGAGAAGGTGGTGAA probe-miR-198 miR-198 CCTATCTCCCCTCTGGACC
probe-miR-199a miR-199a GAACAGGTAGTCTGAACACTGGG probe-miR-
miR-199a* AACCAATGTGCAGACTACTGTA 199a* probe-miR-199b miR-199b
GAACAGATAGTCTAAACACTGGG probe-miR-19a miR-19a
TCAGTTTTGCATAGATTTGCACA probe-miR-19b miR-19b
TCAGTTTTGCATGGATTTGCACA probe-miR-200a miR-200a
ACATCGTTACCAGACAGTGTTA probe-miR- miR-200a* TCCAGCACTGTCCGGTAAGATG
200a* probe-miR-200b miR-200b GTCATCATTACCAGGCAGTATTA
probe-miR-200c miR-200c CCATCATTACCCGGCAGTATTA probe-miR-202
miR-202 TTTTCCCATGCCCTATACCTCT probe-miR-202* miR-202*
AAAGAAGTATATGCATAGGAAA probe-miR-203 miR-203 CTAGTGGTCCTAAACATTTCAC
probe-miR-204 miR-204 AGGCATAGGATGACAAAGGGAA probe-miR-205 miR-205
CAGACTCCGGTGGAATGAAGGA probe-miR-206 miR-206 CCACACACTTCCTTACATTCCA
probe-miR-208 miR-208 ACAAGCTTTTTGCTCGTCTTAT probe-miR-20a miR-20a
CTACCTGCACTATAAGCACTTTA probe-miR-20b miR-20b
CTACCTGCACTATGAGCACTTTG probe-miR-21 miR-21 TCAACATCAGTCTGATAAGCTA
probe-miR-210 miR-210 TCAGCCGCTGTCACACGCACAG probe-miR-211 miR-211
AGGCGAAGGATGACAAAGGGAA probe-miR-212 miR-212 GGCCGTGACTGGAGACTGTTA
probe-miR-213 miR-213 GGTACAATCAACGGTCGATGGT probe-miR-214 miR-214
CTGCCTGTCTGTGCCTGCTGT probe-miR-215 miR-215 GTCTGTCAATTCATAGGTCAT
probe-miR-216 miR-216 CACAGTTGCCAGCTGAGATTA
probe-miR-217 miR-217 ATCCAATCAGTTCCTGATGCAGTA probe-miR-218
miR-218 ACATGGTTAGATCAAGCACAA probe-miR-219 miR-219
AGAATTGCGTTTGGACAATCA probe-miR-22 miR-22 ACAGTTCTTCAACTGGCAGCTT
probe-miR-220 miR-220 AAAGTGTCAGATACGGTGTGG probe-miR-221 miR-221
GAAACCCAGCAGACAATGTAGCT probe-miR-222 miR-222
GAGACCCAGTAGCCAGATGTAGCT probe-miR-223 miR-223
GGGGTATTTGACAAACTGACA probe-miR-224 miR-224 TAAACGGAACCACTAGTGACTTG
probe-miR-23a miR-23a GGAAATCCCTGGCAATGTGAT probe-miR-23b miR-23b
GGTAATCCCTGGCAATGTGAT probe-miR-24 miR-24 CTGTTCCTGCTGAACTGAGCCA
probe-miR-25 miR-25 TCAGACCGAGACAAGTGCAATG probe-miR-26a miR-26a
GCCTATCCTGGATTACTTGAA probe-miR-26b miR-26b AACCTATCCTGAATTACTTGAA
probe-miR-27a miR-27a GCGGAACTTAGCCACTGTGAA probe-miR-27b miR-27b
GCAGAACTTAGCCACTGTGAA probe-miR-28 miR-28 CTCAATAGACTGTGAGCTCCTT
probe-miR-296 miR-296 ACAGGATTGAGGGGGGGCCCT probe-miR-299- miR-299-
AAGCGGTTTACCATCCCACATA 3p 3p probe-miR-299- miR-299-
ATGTATGTGGGACGGTAAACCA 5p 5p probe-miR-29a miR-29a
AACCGATTTCAGATGGTGCTA probe-miR-29b miR-29b AACACTGATTTCAAATGGTGCTA
probe-miR-29c miR-29c ACCGATTTCAAATGGTGCTA probe-miR-301 miR-301
GCTTTGACAATACTATTGCACTG probe-miR-302a miR-302a
TCACCAAAACATGGAAGCACTTA probe-miR- miR-302a* AAAGCAAGTACATCCACGTTTA
302a probe-miR-302b miR-302b CTACTAAAACATGGAAGCACTTA probe-miR-
miR-302b* AGAAAGCACTTCCATGTTAAAGT 302b* probe-miR-302c miR-302c
CCACTGAAACATGGAAGCACTTA probe-miR- miR-302c* CAGCAGGTACCCCCATGTTAAA
302c* probe-miR-302d miR-302d ACACTCAAACATGGAAGCACTTA
probe-miR-30a- miR-30a- GCTGCAAACATCCGACTGAAAG 3p 3p probe-miR-30a-
miR-30a- CTTCCAGTCGAGGATGTTTACA 5p 5p probe-miR-30b miR-30b
AGCTGAGTGTAGGATGTTTACA probe-miR-30c miR-30c
GCTGAGAGTGTAGGATGTTTACA probe-miR-30d miR-30d
CTTCCAGTCGGGGATGTTTACA probe-miR-30e- miR-30e-
GCTGTAAACATCCGACTGAAAG 3p 3p probe-miR-30e- miR-30e-
TCCAGTCAAGGATGTTTACA 5p 5p probe-miR-31 miR-31
CAGCTATGCCAGCATCTTGCC probe-miR-32 miR-32 GCAACTTAGTAATGTGCAATA
probe-miR-320 miR-320 TTCGCCCTCTCAACCCAGCTTTT probe-miR-323 miR-323
AGAGGTCGACCGTGTAATGTGC probe-miR-324-3p miR-324-
CCAGCAGCACCTGGGGCAGTGG 3p 3p probe-miR-324- miR-324-
ACACCAATGCCCTAGGGGATGCG 5p 5p probe-miR-325 miR-325
ACACTTACTGGACACCTACTAGG probe-miR-326 miR-326 CTGGAGGAAGGGCCCAGAGG
probe-miR-328 miR-328 ACGGAAGGGCAGAGAGGGCCAG probe-miR-329 miR-329
AAAGAGGTTAACCAGGTGTGTT probe-miR-33 miR-33 CAATGCAACTACAATGCAC
probe-miR-330 miR-330 TCTCTGCAGGCCGTGTGCTTTGC probe-miR-331 miR-331
TTCTAGGATAGGCCCAGGGGC probe-miR-335 miR-335 ACATTTTTCGTTATTGCTCTTGA
probe-miR-337 miR-337 AAAGGCATCATATAGGAGCTGGA probe-miR-338 miR-338
TCAACAAAATCACTGATGCTGGA probe-miR-339 miR-339 TGAGCTCCTGGAGGACAGGGA
probe-miR-33b miR-33b TGCAATGCAACAGCAATGCAC probe-miR-340 miR-340
GGCTATAAAGTAACTGAGACGGA probe-miR-342 miR-342
GACGGGTGCGATTTCTGTGTGAGA probe-miR-345 miR-345
GCCCTGGACTAGGAGTCAGCA probe-miR-346 miR-346 AGAGGCAGGCATGCGGGCAGACA
probe-miR-34a miR-34a AACAACCAGCTAAGACACTGCCA probe-miR-34b miR-34b
CAATCAGCTAATGACACTGCCTA probe-miR-34c miR-34c
GCAATCAGCTAACTACACTGCCT probe-miR-361 miR-361
GTACCCCTGGAGATTCTGATAA probe-miR-362 miR-362
CTCACACCTAGGTTCCAAGGATT probe-miR-363 miR-363
TTACAGATGGATACCGTGCAAT probe-miR-363* miR-363*
AAATTGCATCGTGATCCACCCG probe-miR-365 miR-365 ATAAGGATTTTTAGGGGCATTA
probe-miR-367 miR-367 TCACCATTGCTAAAGTGCAATT probe-miR-368 miR-368
AAACGTGGAATTTCCTCTATGT probe-miR-369- miR-369-
AAAGATCAACCATGTATTATT 3p 3p probe-miR-369- miR-369-
GCGAATATAACACGGTCGATCT 5p 5p probe-miR-370 miR-370
CCAGGTTCCACCCCAGCAGGC probe-miR-371 miR-371 ACACTCAAAAGATGGCGGCAC
probe-miR-372 miR-372 ACGCTCAAATGTCGCAGCACTTT probe-miR-373 miR-373
ACACCCCAAAATCGAAGCACTTC probe-miR-373* miR-373*
GGAAAGCGCCCCCATTTTGAGT probe-miR-374 miR-374 CACTTATCAGGTTGTATTATAA
probe-miR-375 miR-375 TCACGCGAGCCGAACGAACAAA probe-miR-376a
miR-376a ACGTGGATTTTCCTCTATGAT probe-miR- miR-376a*
CTCATAGAAGGAGAATCTACC 376a* probe-miR-376b miR-376b
AACATGGATTTTCCTCTATGAT probe-miR-377 miR-377 ACAAAAGTTGCCTTTGTGTGAT
probe-miR-378 miR-378 ACACAGGACCTGGAGTCAGGAG probe-miR-379 miR-379
TACGTTCCATAGTCTACCA probe-miR-380- miR-380- AAGATGTGGACCATATTACATA
3p 3p probe-miR-380- miR-380- GCGCATGTTCTATGGTCAACCA 5p 5p
probe-miR-381 miR-381 ACAGAGAGCTTGCCCTTGTATA probe-miR-382 miR-382
CGAATCCACCACGAACAACTTC probe-miR-383 miR-383 AGCCACAATCACCTTCTGATCT
probe-miR-384 miR-384 TATGAACAATTTCTAGGAAT probe-miR-409- miR-409-
AGGGGTTCACCGAGCAACATTCG 3p 3p probe-miR-409- miR-409-
TGCAAAGTTGCTCGGGTAACCT 5p 5p probe-miR-410 miR-410
AACAGGCCATCTGTGTTATATT probe-miR-411 miR-411 CGTACGCTATACGGTCTACTA
probe-miR-412 miR-412 ACGGCTAGTGGACCAGGTGAAGT probe-miR-421 miR-421
GCGCCCAATTAATGTCTGTTGAT probe-miR-422a miR-422a
GGCCTTCTGACCCTAAGTCCAG probe-miR-422b miR-422b
GGCCTTCTGACTCCAAGTCCAG probe-miR-423 miR-423 CTGAGGGGCCTCAGACCGAGCT
probe-miR-424 miR-424 TTCAAAACATGAATTGCTGCTG probe-miR-425 miR-425
GGCGGACACGACATTCCCGAT probe-miR-425- miR-425-
TCAACGGGAGTGATCGTGTCATT 5p 5p probe-miR-429 miR-429
ACGGTTTTACCAGACAGTATTA probe-miR-431 miR-431 TGCATGACGGCCTGCAAGACA
probe-miR-432 miR-432 CCACCCAATGACCTACTCCAAGA nrobe-miR-432*
miR-432* AGACATGGAGGAGCCATCCAG probe-miR-433 miR-433
ACACCGAGGAGCCCATCATGAT probe-miR-448 miR-448 ATGGGACATCCTACATATGCAA
probe-miR-449 miR-449 ACCAGCTAACAATACACTGCCA probe-miR-450 miR-450
TATTAGGAACACATCGCAAAAA probe-miR-451 miR-451
AAACTCAGTAATGGTAACGGTTT probe-miR-452 miR-452
GTCTCAGTTTCCTCTGCAAACA probe-miR-452* miR-452*
CTTCTTTGCAGATGAGACTGA probe-miR-453 miR-453
CGAACTCACCACGGACAACCTC
probe-miR-455 miR-455 CGATGTAGTCCAAAGGCACATA probe-miR-483 miR-483
AGAAGACGGGAGGAGAGGAGTGA probe-miR-484 miR-484
ATCGGGAGGGGACTGAGCCTGA probe-miR-485- miR-485- AGAGGAGAGCCGTGTATGAC
3p 3p probe-miR-485- miR-485- GAATTCATCACGGCCAGCCTCT 5p 5p
probe-miR-486 miR-486 CTCGGGGCAGCTCAGTACAGGA probe-miR-487a
miR-487a AACTGGATGTCCCTGTATGATT probe-miR-487b miR-487b
AAGTGGATGACCCTGTACGATT probe-miR-488 miR-488 TTGAGAGTGCCATTATCTGGG
probe-miR-489 miR-489 GCTGCCGTATATGTGATGTCACT probe-miR-490 miR-490
CAGCATGGAGTCCTCCAGGTTG probe-miR-491 miR-491
TCCTCATGGAAGGGTTCCCCACT probe-miR-492 miR-492
AAGAATCTTGTCCCGCAGGTCCT probe-miR-493 miR-493
AATGAAAGCCTACCATGTACAA probe-miR-493- miR-493-
CTGGCACACAGTAGACCTTCA 3p 3p probe-miR-494 miR-494
AAGAGGTTTCCCGTGTATGTTTCA probe-miR-495 miR-495
AAAGAAGTGCACCATGTTTGTTT probe-miR-496 miR-496 GAGATTGGCCATGTAAT
probe-miR-497 miR-497 ACAAACCACAGTGTGCTGCTG probe-miR-498 miR-498
GAAAAACGCCCCCTGGCTTGAAA probe-miR-499 miR-499
TTAAACATCACTGCAAGTCTTAA probe-miR-500 miR-500
CAGAATCCTTGCCCAGGTGCAT probe-miR-501 miR-501 TCTCACCCAGGGACAAAGGATT
probe-miR-502 miR-502 TAGCACCCAGATAGCAAGGAT probe-miR-503 miR-503
CTGCAGAACTGTTCCCGCTGCTA probe-miR-504 miR-504 ATAGAGTGCAGACCAGGGTCT
probe-miR-505 miR-505 GAGGAAACCAGCAAGTGTTGAC probe-miR-506 miR-506
TCTACTCAGAAGGGTGCCTTA probe-miR-507 miR-507 TTCACTCCAAAAGGTGCAAAA
probe-miR-508 miR-508 TCTACTCCAAAAGGCTACAATCA probe-miR-509 miR-509
TCTACCCACAGACGTACCAATCA probe-miR-510 miR-510
TGTGATTGCCACTCTCCTGAGTA probe-miR-511 miR-511 TGACTGCAGAGCAAAAGACAC
probe-miR-512- miR-512- GACCTCAGCTATGACAGCACTT 3p 3p probe-miR-512-
miR-512- GAAAGTGCCCTCAAGGCTGAGTG 5p 5p probe-miR-513 miR-513
ATAAATGACACCTCCCTGTGAA probe-miR-514 miR-514 CTACTCACAGAAGTGTCAAT
probe-miR-515- miR-515- ACGCTCCAAAAGAAGGCACTC 3p 3p probe-miR-515-
miR-515- CAGAAAGTGCTTTCTTTTGGAGAA 5p 5p probe-miR-516- miR-516-
ACCCTCTGAAAGGAAGCA 3p 3p probe-miR-516- miR-516-
AAAGTGCTTCTTACCTCCAGAT 5p 5p probe-miR-517* miR-517*
AGACAGTGCTTCCATCTAGAGG probe-miR-517a miR-517a
AACACTCTAAAGGGATGCACGAT probe-miR-517b miR-517b
AACACTCTAAAGGGATGCACGA probe-miR-517c miR-517c
ACACTCTAAAAGGATGCACGAT probe-miR-518a miR-518a
TCCAGCAAAGGGAAGCGCTTT probe-miR- miR-518a- AAAGGGCTTCCCTTTGCAGA
518a-2* 2* probe-miR-518b miR-518b ACCTCTAAAGGGGAGCGCTTTG
probe-miR-518c miR-518c CACTCTAAAGAGAAGCGCTTTG probe-miR- miR-518c*
CAGAAAGTGCTTCCCTCCAGAGA 518c* probe-miR-518d miR-518d
GCTCCAAAGGGAAGCGCTTTG probe-miR-518e miR-518e
ACACTCTGAAGGGAAGCGCTTT probe-miR-518f miR-518f
TCCTCTAAAGAGAAGCGCTTT probe-miR- miR-518f* AGAGAAAGTGCTTCCCTCTAGAG
518f* probe-miR-519a miR-519a GTAACACTCTAAAAGGATGCACTTT
probe-miR-519b miR-519b AAACCTCTAAAAGGATGCACTTT probe-miR-519c
miR-519c ATCCTCTAAAAAGATGCACTTT probe-miR-519d miR-519d
ACACTCTAAAGGGAGGCACTTTG probe-miR-519e miR-519e
ACACTCTAAAAGGAGGCACTTT probe-miR- miR-519e* GAAAGTGCTCCCTTTTGGAGAA
519e* probe-miR-520a miR-520a ACAGTCCAAAGGGAAGCACTTT probe-miR-
miR-520a* AGAAAGTACTTCCCTCTGGAG 520a* probe-miR-520b miR-520b
CCCTCTAAAAGGAAGCACTTT probe-miR-520c miR-520c
AACCCTCTAAAAGGAAGCACTTT probe-miR-520d miR-520d
AACCCACCAAAGAGAAGCACTTT probe-miR- miR-520d*
CAGAAAGGGCTTCCCTTTGTAGA 520d* probe-miR-520e miR-520e
CCCTCAAAAAGGAAGCACTTT probe-miR-520f miR-520f
AACCCTCTAAAAGGAAGCACTT probe-miR-520g miR-520g
ACACTCTAAAGGGAAGCACTTTGT probe-miR-520h miR-520h
ACTCTAAAGGGAAGCACTTTGT probe-miR-521 miR-521 ACACTCTAAAGGGAAGTGCGTT
probe-miR-522 miR-522 AACACTCTAAAGGGAACCATTTT probe-miR-523 miR-523
CCCTCTATAGGGAAGCGCGTT probe-miR-524 miR-524 ACTCCAAAGGGAAGCGCCTTC
probe-miR-524* miR-524* GAGAAAGTGCTTCCCTTTGTAG probe-miR-525
miR-525 AGAAAGTGCATCCCTCTGGAG probe-miR-525* miR-525*
GCTCTAAAGGGAAGCGCCTTC probe-miR-526a miR-526a AGAAAGTGCTTCCCTCTAGAG
probe-miR-526b miR-526b AACAGAAAGTGCTTCCCTCAAGAG probe-miR-
miR-526b* GCCTCTAAAAGGAAGCACTTT 526b* probe-miR-526c miR-526c
AACAGAAAGCGCTTCCCTCTAGAG probe-miR-527 miR-527
AGAAAGGGCTTCCCTTTGCAG probe-miR-532 miR-532 ACGGTCCTACACTCAAGGCATG
probe-miR-542- miR-542- TTTCAGTTATCAATCTGTCACA 3p 3p probe-miR-542-
miR-542- CTCGTGACATGATGATCCCCGA 5p 5p probe-miR-544 miR-544
ACTTGCTAAAAATGCAGAAT probe-miR-545 miR-545 CACACAATAAATGTTTGCTGAT
probe-miR-548a miR-548a GCAAAAGTAATTGCCAGTTTTG probe-miR-548b
miR-548b ACAAAAGCAACTGAGGTTCTTG probe-miR-548c miR-548c
GCAAAAGTAATTGAGATTTTTG probe-miR-548d miR-548d
GCAAAAGAAACTGTGGTTTTTG probe-miR-549 miR-549 AGAGCTCATCCATAGTTGTCA
probe-miR-550 miR-550 ATGTGCCTGAGGGAGTAAGACA probe-miR-551a
miR-551a TGGAAACCAAGAGTGGGTCGC probe-miR-552 miR-552
TTGTCTAACCAGTCACCTGTT probe-miR-553 miR-553 AAAACAAAATCTCACCGTTTT
probe-miR-554 miR-554 ACTGGCTGAGTCAGGACTAGC probe-miR-555 miR-555
ATCAGAGGTTCAGCTTACCCT probe-miR-556 miR-556 CATATTACAATGAGCTCATC
probe-miR-557 miR-557 AGACAAGGCCCACCCGTGCAAAC probe-miR-558 miR-558
ATTTTGGTACAGCAGCTCA probe-miR-559 miR-559 TTTTGGTGCATATTTACTTTA
probe-miR-560 miR-560 GGCGGCCGGCCGGCGCACGC probe-miR-561 miR-561
ACTTCAAGGATCTTAAACTTTG probe-miR-562 miR-562 GCAAATGGTACAGCTACTTT
probe-miR-563 miR-563 GGGAAACGTATGTCAACCT probe-miR-564 miR-564
GCCTGCTGACACCGTGCCT probe-miR-565 miR-565 AAACAGACATCGCGAGCCAGCC
probe-miR-566 miR-566 GTTGGGATCACAGGCGCCC probe-miR-567 miR-567
GTTCTGTCCTGGAAGAACATACT probe-miR-568 miR-568 GTGTGTATACATTTATACAT
probe-miR-569 miR-569 ACTTTCCAGGATTCATTAACT probe-miR-570 miR-570
TGCAAAGGTAATTGCTGTTTTC probe-miR-571 miR-571 CTCACTCAGATGGCCAACTCA
probe-miR-572 miR-572 TGGGCCACCGCCGAGCGGAC probe-miR-573 miR-573
CTGATCAGTTACACATCACTTCAG
probe-miR-574 miR-574 GTGGGTGTGTGCATGAGCGTG probe-miR-575 miR-575
GCTCCTGTCCAACTGGCTC probe-miR-576 miR-576 CAAAGACGTGGAGAAATTAGAAT
probe-miR-577 miR-577 CAGGTACCAATATTTTATCTA probe-miR-578 miR-578
ACAATCCTAGAGCACAAGAAG probe-miR-579 miR-579 ATCGCGGTTTATACCAAATGAAT
probe-miR-580 miR-580 CCTAATGATTCATCATTCTCAA probe-miR-581 miR-581
ACTGATCTAGAGAACACAAGA probe-miR-582 miR-582 AGTAACTGGTTGAACAACTGTAA
probe-miR-583 miR-583 GTAATGGGACCTTCCTCTTTG probe-miR-584 miR-584
CTCAGTCCCAGGCAAACCATAA probe-miR-585 miR-585 TAGCATACAGATACGCCCA
probe-miR-586 miR-586 GGACCTAAAAATACAATGCATA probe-miR-587 miR-587
GTGACTCATCACCTATGGAAA probe-miR-588 miR-588 GTTCTAACCCATTGTGGCCAA
probe-miR-589 miR-589 TCTGGGAACCGGCATTTGTTCTGA probe-miR-590
miR-590 CTGCACTTTTATGAATAAGCTC probe-miR-591 miR-591
ACAATGAGAACCCATGGTCT probe-miR-592 miR-592 ACATCATCGCATATTGACACAA
probe-miR-593 miR-593 GCTGAGCAATGCCTGGCTGGTGCCT probe-miR-594
miR-594 AAAGTCACAGGCCACCCCAGATGGG probe-miR-595 miR-595
AGACACACCACGGCACACTTC probe-miR-596 miR-596 CCCGAGGAGCCGGGCAGGCTT
probe-miR-597 miR-597 ACAGTGGTCATCGAGTGACACA probe-miR-598 miR-598
TGACGATGACAACGATGACGTA probe-miR-599 miR-599 GTTTGATAAACTGACACAAC
probe-miR-600 miR-600 GAGCAAGGCTCTTGTCTGTAAGT probe-miR-601 miR-601
CTCCTCCAACAATCCTAGACCA probe-miR-602 miR-602
GGGCCGCAGCTGTCGCCCGTGTC probe-miR-603 miR-603
GCAAAAGTAATTGCAGTGTGTG probe-miR-604 miR-604 GTCCTGAATTCCGCAGCCT
probe-miR-605 miR-605 AGGAGAAGGCACCATGGGATTTA probe-miR-606 miR-606
ATCTTTGATTTTCAGTAGTTT probe-miR-607 miR-607 GTTATAGATCTGGATTTGAAC
probe-miR-608 miR-608 ACGGAGCTGTCCCAACACCACCCCT probe-miR-609
miR-609 AGAGATGAGAGAAACACCCT probe-miR-610 miR-610
TCCCAGCACACATTTAGCTCA probe-miR-611 miR-611 GTCAGACCCCGAGGGGTCCTCGC
probe-miR-612 miR-612 AAGGAGCTCAGAAGCCCTGCCCAGC probe-miR-613
miR-613 GGCAAAGAAGGAACATTCCT probe-miR-614 miR-614
CCACCTGGCAAGAACAGGCGTTC probe-miR-615 miR-615 AGAGGGAGACCCAGGCTCGGA
probe-miR-616 miR-616 AAGTCACTGAAGGGTTTTGAGT probe-miR-617 miR-617
GCCACCTTCAAATGGGAAGTCT probe-miR-618 miR-618
ACTCAGAAGGACAAGTAGAGTTT probe-miR-619 miR-619
ACTGGGCACAAACATGTCCAGGTC probe-miR-620 miR-620 ATTTCTATATCTATCTCCAT
probe-miR-621 miR-621 AGGTAAGCGCTGTTGCTAGCC probe-miR-622 miR-622
GCTCCAACCTCAGCAGACTGT probe-miR-623 miR-623 ACCCAACAGCCCCTGCAAGGGAT
probe-miR-624 miR-624 TGAACACAAGGTACTGGTACTA probe-miR-625 miR-625
AGGACTATAGAACTTTCCCCCT probe-miR-626 miR-626 AAGACATTTTCAGACAGCT
probe-miR-627 miR-627 TCCTCTTTTCTTAGAGACTCAC probe-miR-628 miR-628
CGACTGCCACTCTTACTAGA probe-miR-629 miR-629 GCTGGGCTTACGTTGGGAGAAC
probe-miR-630 miR-630 ACCTTCCCTGGTACAGAATACT probe-miR-631 miR-631
GCTGAGGTCTGGGCCAGGTCT probe-miR-632 miR-632 TCCCACAGGAAGCAGACAC
probe-miR-633 miR-633 TTTATTGTGGTAGATACTATTAG probe-miR-634 miR-634
GTCCAAAGTTGGGGTGCTGGTT probe-miR-635 miR-635
GGACATTGTTTCAGTGCCCAAGT probe-miR-636 miR-636
CTGCGGGCGGGACGAGCAAGCACA probe-miR-637 miR-637
ACGCAGAGCCCGAAAGCCCCCAGT probe-miR-638 miR-638
AGGCCGCCACCCGCCCGCGATCCCT probe-miR-639 miR-639
ACAGCGCTCGCAACCGCAGCGAT probe-miR-640 miR-640 AGAGGCAGGTTCCTGGATCAT
probe-miR-641 miR-641 GAGGTGACTCTATCCTATGTCTTT probe-miR-642
miR-642 CAAGACACATTTGGAGAGGGAC probe-miR-643 miR-643
CTACCTGAGCTAGCATACAAGT probe-miR-644 miR-644 GCTCTAAGAAAGCCACACT
probe-miR-645 miR-645 TCAGCAGTACCAGCCTAGA probe-miR-646 miR-646
GCCTCAGAGGCAGCTGCTT probe-miR-647 miR-647 GAAGGAAGTGAGTGCAGCCAC
probe-miR-648 miR-648 ACCAGTGCCCTGCACACTT probe-miR-649 miR-649
GACTCTTGAACAACACAGGTTT probe-miR-650 miR-650 GTCCTGAGAGCGCTGCCTCCT
probe-miR-651 miR-651 CAAAAGTCAAGCTTATCCTAAA probe-miR-652 miR-652
TGCACAACCCTAGTGGCGCCATT probe-miR-653 miR-653 GTTCAGTAGAGATTGTTTCAA
probe-miR-654 miR-654 GCACATGTTCTGCGGCCCACCA probe-miR-655 miR-655
AAAGAGGTTAACCATGTATTAT probe-miR-656 miR-656 AGAGGTTGACTGTATAATATT
probe-miR-657 miR-657 CCTAGAGAGGGTGAGAACCTGCC probe-miR-658 miR-658
ACCAACGGACCTACTTCCCTCCGCC probe-miR-659 miR-659
TGGGGACCCTCCCTGAACCAAG probe-miR-660 miR-660 CAACTCCGATATGCAATGGGTA
probe-miR-661 miR-661 ACGCGCAGGCCAGAGACCCAGGCA probe-miR-662
miR-662 CTGCTGGGCCACAACGTGGGA probe-miR-663 miR-663
GCGGTCCCGCGGCGCCCCGCCT probe-miR-7 miR-7 CAACAAAATCACTAGTCTTCCA
probe-miR-9 miR-9 TCATACAGCTAGATAACCAAAGA probe-miR-9* miR-9*
ACTTTCGGTTATCTAGCTTTA probe-miR-92 miR-92 CAGGCCGGGACAAGTGCAATA
probe-miR-93 miR-93 CTACCTGCACGAACAGCACTTT probe-miR-95 miR-95
TGCTCAATAAATACCCGTTGAA probe-miR-96 miR-96 GCAAAAATGTGCTAGTGCCAAA
probe-miR-98 miR-98 AACAATACAACTTACTACCTCA probe-miR-99a miR-99a
CACAAGATCGGATCTACGGGTT probe-miR-99b miR-99b
CGCAAGGTCGGTTCTACGGGTG
43. The biochip according to claim 40, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to determine the physiological and/or pathological
condition of the subject after being administrated a test
sample.
44. The biochip according to claims 43, characterized in that the
biochip are useful for screening the test sample for the activities
on the prevention and/or treatment of diseases.
45. The biochip according to claim 40, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to diagnose and/or differentially diagnose the diseases
of the subject.
46. The biochip according to claim 40, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to evaluate the effectiveness of treating the diseases
of the subject.
47. The biochip according to claim 40, characterized in that said
evaluating the physiological and/or pathological condition of a
subject is to predict the occurrence of diseases of the
subject.
48. The biochip according to claim 47, characterized in that the
occurrence of diseases is the occurrence of complications and/or
the relapse of malignant diseases.
49. The biochip according to claim 44, characterized in that the
diseases include various tumors, various acute and chronic
infectious diseases e.g. viral diseases such as viral influenza,
viral hepatitis, AIDS, SARS, bacterial diseases such as
tuberculosis, bacterial pneumonia, and other acute and chronic
infectious diseases caused by various pathogenic microorganisms;
other acute and chronic diseases such as diseases of respiratory
system, diseases of immune system, diseases of blood and
hematopoietic system, diseases of circulatory system such as
cardio-cerebrovascular disease, metabolic diseases of endocrine
system, diseases of digestive system, diseases of nervous system,
diseases of urinary system, diseases of reproductive system and
diseases of locomotor system.
50. The biochip according to claim 40, characterized, in that the
biochip are useful for detecting the subject for prohibited
drugs-taking.
51. The chip according to claim 40, characterized in that the
serum/plasma the subject is from living bodies, tissues, organs
and/or corpuses of the subject.
Description
TECHNICAL FIELD
[0001] The present invention relates to microRNAs and uses thereof,
more specifically, to serum/plasma microRNAs and the uses of
serum/plasma microRNAs for diagnosis and differential diagnosis of
diseases, prediction of complication occurrence and malignant
disease relapse, evaluation of therapeutic effects, screening of
pharmaceutical active ingredients, assessment of drug efficacy,
forensic authentication and prohibited drug inspection and the
like.
BACKGROUND ART
[0002] To locate and precisely detect disease markers has already
been the important precondition for the diagnosis and treatment of
various clinical diseases including various tumors; various
acute/chronic infectious diseases, e.g. viral diseases such as
viral influenza, viral hepatitis, AIDS, SARS, bacterial diseases
such as tuberculosis, bacterial pneumonia, and other acute/chronic
infectious diseases caused by various pathogenic microorganisms;
other acute/chronic diseases such as diseases of respiratory
system, diseases of immune system, diseases of blood and
hematopoietic system, diseases of circulatory system such as
cardio-cerebrovascular diseases, metabolic diseases of endocrine
system, diseases of digestive system, diseases of nervous system,
diseases of urinary system, diseases of reproductive system and
diseases of locomotor system. Although more and more disease
markers have been found and utilized in general survey and
diagnosis of clinical diseases as well as monitoring and
controlling of therapeutic effects, their clinical application
effects are obviously insufficient. For instance, tumor marker,
e.g. alphafetoprotein, lactic dehydrogenase and carcinoembryonic
antigen have been widely used in clinic. But these disease markers
are far from meeting the needs of early diagnosis for cancer for
the following two main reasons: (1) the sensitivity and specificity
for the above-mentioned disease markers are relatively low, thus
their detection results cannot be used as a diagnostic indicator of
disease; (2) the early diagnosis rate of disease shall be
positively correlative with the therapeutic effects. However, it is
difficult for any of the aforesaid disease markers to meet such
requirements for early diagnosis. Take cancer for example, the
specificity of tumor differentiation is too high, the integrated
sensitivity of tumor is relatively low, the samples sent to be
detected are difficult to be repeatedly taken and the conditions to
meet the preservation requirements for samples are too exacting,
meanwhile, the cost is very high, thus under existing technology
the spreading and use of the tumor markers available are hard to
realize. The inherent defects of some traditional medical means
such as biopsy, for example, incorrect material-extraction
position, the inadequacy of sample materials for histocytes and
human inexperience, etc., will all lead to misdiagnosis. Although
other techniques such as imaging technique have been widely used
for examination and diagnosis of diseases, there exists
considerable limitation on the determination for disease degree.
Consequently, it is very necessary to find out a maker for disease
detection which is novel, sensitive and convenient to use and can
also overcome the defects of existing markers as mentioned
above.
[0003] MicroRNAs are defined as a kind of non-coding
single-stranded small RNA moleculars of approximately from 19 to 23
nucleotides in length. They are highly conservative in evolution;
and are closely related to many normal physiological activities of
animals such as development process, tissue differentiation, cell
apoptosis and energy metabolism; in addition, bear close relation
with the occurrence and development of many diseases. Recent
studies show that the expression levels of several microRNAs in
chronic lymphocytic leukemia and Burkitt lymphoma are on average
down-regulated to various extents; and that by analyzing and
comparing the expressions of microRNAs in tissues of human lung
cancer and human breast cancer, the expression levels of several
tissue specific microRNAs have changed relative to normal tissues.
Some studies demonstrate that microRNAs affect the occurrence and
development of cardio-cerebrovascular diseases such as myocardial
hypertrophy, heart failure, atherosclerosis, and are closely
relative to metabolic diseases such as Diabetes II. These
experimental results indicate that there exists inevitable
connection between the expression and specificity changes of
microRNAs and the occurrence and development of diseases.
[0004] For the unimaginable important role microRNAs played in the
regulation of expression after gene transcription, microRNAs have
some associations with diseases. First of all, the changes of
microRNAs may be the cause of diseases. This is because both the
inhibitor and the promoter of diseases may be target sites for
microRNAs. If the expression of microRNA itself is disturbed, e.g.,
the expression level of microRNA which is originally to inhibit
disease promoters decreases or the expression level of microRNA
which is to inhibit disease inhibitor increases, its end results
will both lead to changes in the expression of downstream genes and
the overall disorder of some pathways, further inducing the
occurrence of diseases. Secondly, the changes of microRNAs may also
result from diseases. This is because, when a kind of disease such
as cancer occurs, it will lead to the loss of chromosome segments,
gene mutation or rapid amplification of chromosome segments;
moreover, if the microRNAs happen to locate in the changing
segment, then their expression level will extremely significantly
change. Therefore, in theory, microRNAs can be completely regarded
as a kind of new disease markers, the specificity changes of which
inevitably correlate with the occurrence and development of
diseases. Meanwhile, microRNA can also be used as a potential drug
target, and it may greatly alleviate the occurrence and development
of diseases by inhibiting the up-regulated microRNAs and
overexpressedly down-regulated microRNAs in the course of a
disease.
[0005] The inventor has carried out the research in the relevant
fields of using microRNAs as disease markers, for instance,
choosing colonic carcinoma which ranks forth in the incidence of
malignant tumor as the research object. The research suggests that,
during the process of colon benign polyps developing into malignant
tumor, some microRNAs exhibit specificity changes, thereby a more
sensitive and accurate method for the early diagnosis of colonic
carcinoma having been set up through detecting the specific changes
in microRNAs. However, since the sampling for tissue specimen is
not easy, the wide application of this method in clinics is
limited.
DETAILED DESCRIPTION OF THE INVENTION
[0006] The inventor focuses the research on the blood which is
relatively easy to obtain and even can be collected via routine
physical examination. Blood will circulate to all tissues in body
and convey nutrients to cells whilst scavenging waste materials;
therefore, blood is able to reflect the physiological pathology of
the whole organism and its detection results is an indicator of
human health. It is known that in serum/plasma there are many kinds
of proteins such as total protein, albumin and globulin, many kinds
of lipids such as HDL cholesterol and triglycerides, many kinds of
carbohydrates, pigments, electrolytes, inorganic salts, and many
kinds of enzymes such as amylase, alkaline phosphatase, acid
phosphatase, cholinesterase and aldolase; moreover, there also
exist many kinds of signaling molecules such as cytokines and
hormone from tissues and organs in whole body. Currently, disease
diagnosis is only limited to the above-mentioned biochemical
indexes in serum/plasma, and no report is available regarding
microRNAs in serum/plasma. It traditionally believed that there is
no microRNA in serum/plasma, and that, if any, it will be rapidly
degraded by RNase into small molecule segments and hence cannot be
detected. However, microRNAs, consisting of from 19 to 23
nucleotides, possess specificity and relative stability in
structure and hence are very likely present in serum/plasma.
Meanwhile, since microRNAs are a new type of disease markers, it is
anticipated that by studying whether or not microRNAs are present
in serum/plasma, whether or not they can be detected and the
connection between microRNAs and diseases, a new technology is
established for the early disease diagnosis, disease identification
as well as monitoring and controlling of course of diseases,
prediction of malignant disease relapse and prognosis and
complication occurrence, assessment of drug efficacy, guide of
medication, individualized treatment, screening of active
ingredients of Chinese Traditional Medicines, population taxonomy,
etc., by use of the microRNAs stably existing in serum/plasma as
well as their specificity changes.
[0007] The present invention provides a combination of microRNAs
for evaluating physiological and/or pathological condition in a
subject, wherein the combination comprises all detectable microRNAs
stably existing in the serum/plasma of the subject.
[0008] The present invention further provides a method for
evaluating physiological and/or pathological condition in a
subject, wherein the method comprises determining all detectable
microRNAs stably existing in the serum/plasma of the subject.
[0009] In the above-mentioned combination or method, all detectable
microRNAs stably existing in serum/plasma of a subject may be all
mature microRNAs in human serum/plasma, specifically include
let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i,
miR-1, miR-100, miR-101, miR-103, miR-105, miR-106a, miR-106b,
miR-107, miR-10a, miR-10b, miR-122a, miR-124a, miR-125a, miR-125b,
miR-126, miR-126*, miR-127, miR-128a, miR-128b, miR-129, miR-130a,
miR-130b, miR-132, miR-133a, miR-133b, miR-134, miR-135a, miR-135b,
miR-136, miR-137, miR-138, miR-139, miR-140, miR-141, miR-142-3p,
miR-142-5p, miR-143, miR-144, miR-145, miR-146a, miR-146b, miR-147,
miR-148a, miR-148b, miR-149, miR-150, miR-151, miR-152, miR-153,
miR-154, miR-154*, miR-155, miR-15a, miR-15b, miR-16, miR-17-3p,
miR-17-5p, miR-181a, miR-181b, miR-181c, miR-181d, miR-182,
miR-182*, miR-183, miR-184, miR-185, miR-186, miR-187, miR-188,
miR-189, miR-18a, miR-18a*, miR-18b, miR-190, miR-191, miR-191*,
miR-192, miR-193a, miR-193b, miR-194, miR-195, miR-196a, miR-196b,
miR-197, miR-198, miR-199a, miR-199a*, miR-199b, miR-19a, miR-19b,
miR-200a, miR-200a*, miR-200b, miR-200c, miR-202, miR-202*,
miR-203, miR-204, miR-205, miR-206, miR-208, miR-20a, miR-20b,
miR-21, miR-210, miR-211, miR-212, miR-213, miR-214, miR-215
miR-216, miR-217, miR-218, miR-219, miR-22, miR-220, miR-221
miR-222, miR-223, miR-224, miR-23a, miR-23b, miR-24, miR-25,
miR-26a, miR-26b, miR-27a, miR-27b, miR-28, miR-296, miR-299-3p,
miR-299-5p, miR-29a, miR-29b, miR-29c, miR-301, miR-302a,
miR-302a*, miR-302b, miR-302b*, miR-302c, miR-302c*, miR-302d,
miR-30a-3p, miR-30a-5p, miR-30b, miR-30e, miR-30d, miR-30e-3p,
miR-30e-5p, miR-32, miR-320, miR-323, miR-324-3p, miR-324-5p,
miR-325, miR-326, miR-328, miR-329, miR-33, miR-330, miR-331,
miR-335, miR-337, miR-338, miR-339, miR-33b, miR-340, miR-342,
miR-345, miR-346, miR-34a, miR-34b, miR-34c, miR-361, miR-362,
miR-363, miR-363*, miR-365, miR-367, miR-368, miR-369-3p,
miR-369-5p, miR-370, miR-371, miR-372, miR-373, miR-373*, miR-374,
miR-375, miR-376a, miR-376a*, miR-376b, miR-377, miR-378, miR-379,
miR-380-3p, miR-380-5p, miR-381, miR-382, miR-383, miR-384,
miR-409-3p, miR-409-5p, miR-410, miR-411, miR-412, miR-421,
miR-422a, miR-422b, miR-423, miR-424, miR-425, miR-425-5p, miR-429,
miR-431, miR-432, miR-432*, miR-433, miR-448, miR-449, miR-450,
miR-451, miR-452, miR-452*, miR-453, miR-455, miR-483, miR-484,
miR-485-3p, miR-485-5p, miR-486, miR-487a, miR-487b, miR-488,
miR-489, miR-490, miR-491, miR-492, miR-493, miR-493-3p, miR-494,
miR-495, miR-496, miR-497, miR-498, miR-499, miR-500, miR-501,
miR-502, miR-503, miR-504, miR-505, miR-506, miR-507, miR-508,
miR-509, miR-510, miR-511, miR-512-3p, miR-512-5p, miR-513,
miR-514, miR-515-3p, miR-515-5p, miR-516-3p, miR-516-5p, miR-517*,
miR-517a, miR-517b, miR-517c, miR-518a, miR-518a-2*, miR-518b,
miR-518c, miR-518c*, miR-518d, miR-518e, miR-518f, miR-518f*,
miR-519a, miR-519b, miR-519c, miR-519d, miR-519e, miR-519e*,
miR-520a, miR-520a*, miR-520b, miR-520c, miR-520d, miR-520d*,
miR-520e, miR-520f, miR-520g, miR-520h, miR-521, miR-522, miR-523,
miR-524, miR-524*, miR-525, miR-525*, miR-526a, miR-526b,
miR-526b*, miR-526c, miR-527, miR-532, miR-542-3p, miR-542-5p,
miR-544, miR-545, miR-548a, miR-548b, miR-548c, miR-548d, miR-549,
miR-550, miR-551a, miR-552, miR-553, miR-554, miR-555, miR-556,
miR-557, miR-558, miR-559, miR-560, miR-561, miR-562, miR-563,
miR-564, miR-565, miR-566, miR-567, miR-568, miR-569, miR-570,
miR-571, miR-572, miR-573, miR-574, miR-575, miR-576, miR-577,
miR-578, miR-579, miR-580, miR-581, miR-582, miR-583, miR-584,
miR-585, miR-586, miR-587, miR-588, miR-589, miR-590, miR-591,
miR-592, miR-593, miR-594, miR-595, miR-596, miR-597, miR-598,
miR-599, miR-600, miR-601, miR-602, miR-603, miR-604, miR-605,
miR-606, miR-607, miR-608, miR-609, miR-610, miR-611, miR-612,
miR-613, miR-614, miR-615, miR-616, miR-617, miR-618, miR-619,
miR-620, miR-621, miR-622, miR-623, miR-624, miR-625, miR-626,
miR-627, miR-628, miR-629, miR-630, miR-631, miR-632, miR-633,
miR-634, miR-635, miR-636, miR-637, miR-638, miR-639, miR-640,
miR-641, miR-642, miR-643, miR-644, miR-645, miR-646, miR-647,
miR-648, miR-649, miR-650, miR-651, miR-652, miR-653, miR-654,
miR-655, miR-656, miR-657, miR-658, miR-659, miR-660, miR-661,
miR-662, miR-663, miR-7, miR-9, miR-9*, miR-92, miR-93, miR-95,
miR-96, miR-98, miR-99 and miR-99b.
[0010] The aforesaid method for determining all detectable
microRNAs stably existing in serum/plasma of a subject is one or
more selected from the group consisting of RT-PCR method, Real-time
PCR method, Northern blotting method, RNase protection assay,
Solexa sequencing technology and biochip method.
[0011] The aforesaid RT-PCR method includes the following
steps:
[0012] (1) extracting the total RNA from the serum/plasma of a
subject and obtaining cDNA samples by RNA reverse transcription
reaction; or collecting serum/plasma samples from the subject and
conducting reverse transcription reaction with serum/plasma being a
buffer so as to prepare cDNA samples;
[0013] (2) designing a primer by use of microRNAs and conducting
PCR reaction;
[0014] (3) conducting agarose gel electrophoresis of PCR
products;
[0015] (4) observing agarose gel under ultraviolet lamp after EB
staining.
[0016] The aforesaid real-time PCR method includes the following
steps:
[0017] (1) extracting the total RNA from the serum/plasma of a
subject and obtaining cDNA samples by RNA reverse transcription
reaction; or collecting serum/plasma samples from the subject and
conducting reverse transcription reaction with serum/plasma being a
buffer so as to prepare cDNA samples;
[0018] (2) designing a primer by use of microRNAs;
[0019] (3) adding a fluorescent probe to conduct PCR reaction;
[0020] (4) detecting and comparing the variation in levels of
microRNAs in the serum/plasma relative to those of microRNAs in
normal serum/plasma.
[0021] The present invention further provides a kit for evaluating
physiological and/or pathological condition of a subject, wherein
the kit comprises the tools for determining all detectable
microRNAs stably existing in the serum/plasma of the subject. The
kit may comprises the primers of all mature microRNAs in human
serum/plasma, specifically comprises the primers of let-7a, let-7b,
let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, miR-1, miR-100,
miR-101, miR-103, miR-105, miR-106a, miR-106b, miR-107, miR-10a,
miR-10b, miR-122a, miR-124a, miR-125a, miR-125b, miR-126, miR-126*,
miR-127, miR-128a, miR-128b, miR-129, miR-130a, miR-130b, miR-132,
miR-133a, miR-133b, miR-134, miR-135a, miR-135b, miR-136, miR-137,
miR-138, miR-139, miR-140, miR-141, miR-142-3p, miR-142-5p,
miR-143, miR-144, miR-145, miR-146a, miR-146b, miR-147, miR-148a,
miR-148b, miR-149, miR-150, miR-151, miR-152, miR-153, miR-154,
miR-154*, miR-155, miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p,
miR-181a, miR-181b, miR-181c, miR-181d, miR-182, miR-182*, miR-183,
miR-184, miR-185, miR-186, miR-187, miR-188, miR-189, miR-18a,
miR-18a*, miR-18b, miR-190, miR-191, miR-191*, miR-192, miR-193a,
miR-193b, miR-194, miR-195, miR-196a, miR-196b, miR-197, miR-198,
miR-199a, miR-199a*, miR-199b, miR-19a, miR-19b, miR-200a,
miR-200a*, miR-200b, miR-200c, miR-202, miR-202*, miR-203, miR-204,
miR-205, miR-206, miR-208, miR-20a, miR-20b, miR-21, miR-210,
miR-211, miR-212, miR-213, miR-214, miR-215, miR-216, miR-217,
miR-218, miR-219, miR-22, miR-220, miR-221, miR-222, miR-223,
miR-224, miR-23a, miR-23b, miR-24, miR-25, miR-26a, miR-26b,
miR-27a, miR-27b, miR-28, miR-296, miR-299-3p, miR-299-5p, miR-29a,
miR-29b, miR-29c, miR-301, miR-302a, miR-302a*, miR-302b,
miR-302b*, miR-302c, miR-302c*, miR-302d, miR-30a-3p, miR-30a-5p,
miR-30b, miR-30c, miR-30d, miR-30e-3p, miR-30e-5p, miR-32, miR-320,
miR-323, miR-324-3p, miR-324-5p, miR-325, miR-326, miR-328,
miR-329, miR-33, miR-330, miR-331, miR-335, miR-337, miR-338,
miR-339, miR-33b, miR-340, miR-342, miR-345, miR-346, miR-34a,
miR-34b, miR-34c, miR-361, miR-362, miR-363, miR-363*, miR-365,
miR-367, miR-368, miR-369-3p, miR-369-5p, miR-370, miR-371,
miR-372, miR-373, miR-373*, miR-374, miR-375, miR-376a, miR-376a*,
miR-376b, miR-377, miR-378, miR-379, miR-380-3p, miR-380-5p,
miR-381, miR-382, miR-383, miR-384, miR-409-3p, miR-409-5p,
miR-410, miR-411, miR-412, miR-421, miR-422a, miR-422b, miR-423,
miR-424, miR-425, miR-425-5p, miR-429, miR-431, miR-432, miR-432*,
miR-433, miR-448, miR-449, miR-450, miR-451, miR-452, miR-452*,
miR-453, miR-455, miR-483, miR-484, miR-485-3p, miR-485-5p,
miR-486, miR-487a, miR-487b, miR-488, miR-489, miR-490, miR-491,
miR-492, miR-493, miR-493-3p, miR-494, miR-495, miR-496, miR-497,
miR-498, miR-499, miR-500, miR-501, miR-502, miR-503, miR-504,
miR-505, miR-506, miR-507, miR-508, miR-509, miR-510, miR-511,
miR-512-3p, miR-512-5p, miR-513, miR-514, miR-515-3p, miR-515-5p,
miR-516-3p, miR-516-5p, miR-517*, miR-517a, miR-517b, miR-517c,
miR-518a, miR-518a-2*, miR-518b, miR-518c, miR-518c*, miR-518d,
miR-518e, miR-518f, miR-518f*, miR-519a, miR-519b, miR-519c,
miR-519d, miR-519e, miR-519e*, miR-520a, miR-520a*, miR-520b,
miR-520c, miR-520d, miR-520d*, miR-520e, miR-520f, miR-520g,
miR-520h, miR-521, miR-522, miR-523, miR-524, miR-524*, miR-525,
miR-525*, miR-526a, miR-526b, miR-526b* miR-526c, miR-527, miR-532,
miR-542-3p, miR-542-5p, miR-544, miR-545, miR-548a, miR-548b,
miR-548c, miR-548d, miR-549, miR-550, miR-551a, miR-552, miR-553,
miR-554, miR-555, miR-556, miR-557, miR-558, miR-559, miR-560,
miR-561, miR-562, miR-563, miR-564, miR-565, miR-566, miR-567,
miR-568, miR-569, miR-570, miR-571, miR-572, miR-573, miR-574,
miR-575, miR-576, miR-577, miR-578, miR-579, miR-580, miR-581,
miR-582, miR-583, miR-584, miR-585, miR-586, miR-587, miR-588,
miR-589, miR-590, miR-591, miR-592, miR-593, miR-594, miR-595,
miR-596, miR-597, miR-598, miR-599, miR-600, miR-601, miR-602,
miR-603, miR-604, miR-605, miR-606, miR-607, miR-608, miR-609,
miR-610, miR-611, miR-612, miR-613, miR-614, miR-615, miR-616,
miR-617, miR-618, miR-619, miR-620, miR-621, miR-622, miR-623,
miR-624, miR-625, miR-626, miR-627, miR-628, miR-629, miR-630,
miR-631, miR-632, miR-633, miR-634, miR-635, miR-636, miR-637,
miR-638, miR-639, miR-640, miR-641, miR-642, miR-643, miR-644,
miR-645, miR-646, miR-647, miR-648, miR-649, miR-650, miR-651,
miR-652, miR-653, miR-654, miR-655, miR-656, miR-657, miR-658,
miR-659, miR-660, miR-661, miR-662, miR-663, miR-7, miR-9, miR-9*,
miR-92, miR-93, miR-95, miR-96, miR-98, miR-99a and miR-99b.
[0022] The present invention also provides a biochip for evaluating
physiological and/or pathological condition of a subject, wherein
the biochip contains the components for determining all detectable
microRNAs stably existing in the serum/plasma of the subject. The
biochip may also contain the probes for all mature microRNAs in
human serum/plasma. The probes specifically include the probes as
shown in Table 1.
TABLE-US-00001 TABLE 1 Probes of all mature microRNAs in human
serum/plasma Corre- sponding micro- Probes RNAs Sequences of probes
probe-let-7a let-7a AACTATACAACCTACTACCTCA probe-let-7b let-7b
AACCACACAACCTACTACCTCA probe-let-7c let-7c AACCATACAACCTACTACCTCA
probe-let-7d let-7d ACTATGCAACCTACTACCTCT probe-let-7e let-7e
ACTATACAACCTCCTACCTCA probe-let-7f let-7f AACTATACAATCTACTACCTCA
probe-let-7g let-7g ACTGTACAAACTACTACCTCA probe-let-7i let-7i
ACAGCACAAACTACTACCTCA probe-miR-1 miR-1 TACATACTTCTTTACATTCCA
probe-miR-100 miR-100 CACAAGTTCGGATCTACGGGTT probe-miR-101 miR-101
CTTCAGTTATCACAGTACTGTA probe-miR-103 miR-103
TCATAGCCCTGTACAATGCTGCT probe-miR-105 miR-105 ACAGGAGTCTGAGCATTTGA
probe-miR-106a miR-106a GCTACCTGCACTGTAAGCACTTTT probe-miR-106b
miR-106b ATCTGCACTGTCAGCACTTTA probe-miR-107 miR-107
TGATAGCCCTGTACAATGCTGCT probe-miR-10a miR-10a
CACAAATTCGGATCTACAGGGTA probe-miR-10b miR-10b
ACAAATTCGGTTCTACAGGGTA probe-miR-122a miR-122a
ACAAACACCATTGTCACACTCCA probe-miR-124a miR-124a
TGGCATTCACCGCGTGCCTTAA probe-miR-125a miR-125a
CACAGGTTAAAGGGTCTCAGGGA probe-miR-125b miR-125b
TCACAAGTTAGGGTCTCAGGGA probe-miR-126 miR-126 GCATTATTACTCACGGTACGA
probe-miR-126* miR-126* CGCGTACCAAAAGTAATAATG probe-miR-127 miR-127
AGCCAAGCTCAGACGGATCCGA probe-miR-128a miR-128a
AAAAGAGACCGGTTCACTGTGA probe-miR-128b miR-128b
GAAAGAGACCGGTTCACTGTGA probe-miR-129 miR-129 GCAAGCCCAGACCGCAAAAAG
probe-miR-130a miR-130a ATGCCCTTTTAACATTGCACTG probe-miR-130b
miR-130b ATGCCCTTTCATCATTGCACTG probe-miR-132 miR-132
CGACCATGGCTGTAGACTGTTA probe-miR-133a miR-133a
ACAGCTGGTTGAAGGGGACCAA probe-miR-133b miR-133b
TAGCTGGTTGAAGGGGACCAA probe-miR-134 miR-134 CCCTCTGGTCAACCAGTCACA
probe-miR-135a miR-135a TCACATAGGAATAAAAAGCCATA probe-miR-135b
miR-135b CACATAGGAATGAAAAGCCATA probe-miR-136 miR-136
TCCATCATCAAAACAAATGGAGT probe-miR-137 miR-137
CTACGCGTATTCTTAAGCAATA probe-miR-138 miR-138 GATTCACAACACCAGCT
probe-miR-139 miR-139 AGACACGTGCACTGTAGA probe-miR-140 miR-140
CTACCATAGGGTAAAACCACT probe-miR-141 miR-141 CCATCTTTACCAGACAGTGTTA
probe-miR-142- miR-142- TCCATAAAGTAGGAAACACTACA 3p 3p
probe-miR-142- miR-142- GTAGTGCTTTCTACTTTATG 5p 5p probe-miR-143
miR-143 TGAGCTACAGTGCTTCATCTCA probe-miR-144 miR-144
CTAGTACATCATCTATACTGTA probe-miR-145 miR-145
AAGGGATTCCTGGGAAAACTGGAC probe-miR-146a miR-146a
AACCCATGGAATTCAGTTCTCA probe-miR-146b miR-146b
AGCCTATGGAATTCAGTTCTCA probe-miR-147 miR-147 GCAGAAGCATTTCCACACAC
probe-miR-148a miR-148a ACAAAGTTCTGTAGTGCACTGA probe-miR-148b
miR-148b ACAAAGTTCTGTGATGCACTGA probe-miR-149 miR-149
GGAGTGAAGACACGGAGCCAGA probe-miR-150 miR-150 CACTGGTACAAGGGTTGGGAGA
probe-miR-151 miR-151 CCTCAAGGAGCTTCAGTCTAGT probe-miR-152 miR-152
CCCAAGTTCTGTCATGCACTGA probe-miR-153 miR-153 TCACTTTTGTGACTATGCAA
probe-miR-154 miR-154 CGAAGGCAACACGGATAACCTA probe-miR-154*
miR-154* AATAGGTCAACCGTGTATGATT probe-miR-155 miR-155
CCCCTATCACGATTAGCATTAA probe-miR-15a miR-15a CACAAACCATTATGTGCTGCTA
probe-miR-15b miR-15b TGTAAACCATGATGTGCTGCTA probe-miR-16 miR-16
CGCCAATATTTACGTGCTGCTA probe-miR-17- miR-17- ACAAGTGCCTTCACTGCAGT
3p 3p probe-miR-17- miR-17- ACTACCTGCACTGTAAGCACTTTG 5p 5p
probe-miR-181a miR-181a ACTCACCGACAGCGTTGAATGTT probe-miR-181b
miR-181b CCCACCGACAGCAATGAATGTT probe-miR-181c miR-181c
ACTCACCGACAGGTTGAATGTT probe-miR-181d miR-181d
AACCCACCGACAACAATGAATGTT probe-miR-182 miR-182
TGTGAGTTCTACCATTGCCAAA probe-miR-182* miR-182*
TAGTTGGCAAGTCTAGAACCA probe-miR-183 miR-183 CAGTGAATTCTACCAGTGCCATA
probe-miR-184 miR-184 ACCCTTATCAGTTCTCCGTCCA probe-miR-185 miR-185
GAACTGCCTTTCTCTCCA probe-miR-186 miR-186 AAGCCCAAAAGGAGAATTCTTTG
probe-miR-187 miR-187 CGGCTGCAACACAAGACACGA probe-miR-188 miR-188
ACCCTCCACCATGCAAGGGATG probe-miR-189 miR-189
ACTGATATCAGCTCAGTAGGCAC probe-miR-18a miR-18a
TATCTGCACTAGATGCACCTTA probe-miR-18a* miR-18a*
AGAAGGAGCACTTAGGGCAGT probe-miR-18b miR-18b TAACTGCACTAGATGCACCTTA
probe-miR-190 miR-190 ACCTAATATATCAAACATATCA probe-miR-191 miR-191
AGCTGCTTTTGGGATTCCGTTG probe-miR-191* miR-191*
GGGGACGAAATCCAAGCGCAGC probe-miR-192 miR-192 GGCTGTCAATTCATAGGTCAG
probe-miR-193a miR-193a CTGGGACTTTGTAGGCCAGTT probe-miR-193b
miR-193b AAAGCGGGACTTTGAGGGCCAGTT probe-miR-194 miR-194
TCCACATGGAGTTGCTGTTACA probe-miR-195 miR-195 GCCAATATTTCTGTGCTGCTA
probe-miR-196a miR-196a CCAACAACATGAAACTACCTA probe-miR-196b
miR-196b CCAACAACAGGAAACTACCTA probe-miR-197 miR-197
GCTGGGTGGAGAAGGTGGTGAA probe-miR-198 miR-198 CCTATCTCCCCTCTGGACC
probe-miR-199a miR-199a GAACAGGTAGTCTGAACACTGGG probe-miR-199a*
miR-199a* AACCAATGTGCAGACTACTGTA probe-miR-199b miR-199b
GAACAGATAGTCTAAACACTGGG probe-miR-19a miR-19a
TCAGTTTTGCATAGATTTGCACA probe-miR-19b miR-19b
TCAGTTTTGCATGGATTTGCACA probe-miR-200a miR-200a
ACATCGTTACCAGACAGTGTTA probe-miR-200a* miR-200a*
TCCAGCACTGTCCGGTAAGATG probe-miR-200b miR-200b
GTCATCATTACCAGGCAGTATTA probe-miR-200c miR-200c
CCATCATTACCCGGCAGTATTA probe-miR-202 miR-202 TTTTCCCATGCCCTATACCTCT
probe-miR-202* miR-202* AAAGAAGTATATGCATAGGAAA probe-miR-203
miR-203 CTAGTGGTCCTAAACATTTCAC probe-miR-204 miR-204
AGGCATAGGATGACAAAGGGAA probe-miR-205 miR-205 CAGACTCCGGTGGAATGAAGGA
probe-miR-206 miR-206 CCACACACTTCCTTACATTCCA probe-miR-208 miR-208
ACAAGCTTTTTGCTCGTCTTAT probe-miR-20a miR-20a
CTACCTGCACTATAAGCACTTTA probe-miR-20b miR-20b
CTACCTGCACTATGAGCACTTTG probe-miR-21 miR-21 TCAACATCAGTCTGATAAGCTA
probe-miR-210 miR-210 TCAGCCGCTGTCACACGCACAG probe-miR-211 miR-211
AGGCGAAGGATGACAAAGGGAA probe-miR-212 miR-212 GGCCGTGACTGGAGACTGTTA
probe-miR-213 miR-213 GGTACAATCAACGGTCGATGGT probe-miR-214 miR-214
CTGCCTGTCTGTGCCTGCTGT probe-miR-215 miR-215 GTCTGTCAATTCATAGGTCAT
probe-miR-216 miR-216 CACAGTTGCCAGCTGAGATTA
probe-miR-217 miR-217 ATCCAATCAGTTCCTGATGCAGTA probe-miR-218
miR-218 ACATGGTTAGATCAAGCACAA probe-miR-219 miR-219
AGAATTGCGTTTGGACAATCA probe-miR-22 miR-22 ACAGTTCTTCAACTGGCAGCTT
probe-miR-220 miR-220 AAAGTGTCAGATACGGTGTGG probe-miR-221 miR-221
GAAACCCAGCAGACAATGTAGCT probe-miR-222 miR-222
GAGACCCAGTAGCCAGATGTAGCT probe-miR-223 miR-223
GGGGTATTTGACAAACTGACA probe-miR-224 miR-224 TAAACGGAACCACTAGTGACTTG
probe-miR-23a miR-23a GGAAATCCCTGGCAATGTGAT probe-miR-23b miR-23b
GGTAATCCCTGGCAATGTGAT probe-miR-24 miR-24 CTGTTCCTGCTGAACTGAGCCA
probe-miR-25 miR-25 TCAGACCGAGACAAGTGCAATG probe-miR-26a miR-26a
GCCTATCCTGGATTACTTGAA probe-miR-26b miR-26b AACCTATCCTGAATTACTTGAA
probe-miR-27a miR-27a GCGGAACTTAGCCACTGTGAA probe-miR-27b miR-27b
GCAGAACTTAGCCACTGTGAA probe-miR-28 miR-28 CTCAATAGACTGTGAGCTCCTT
probe-miR-296 miR-296 ACAGGATTGAGGGGGGGCCCT probe-miR-299- miR-299-
AAGCGGTTTACCATCCCACATA 3p 3p probe-miR-299- miR-299-
ATGTATGTGGGACGGTAAACCA 5p 5p probe-miR-29a miR-29a
AACCGATTTCAGATGGTGCTA probe-miR-29b miR-29b AACACTGATTTCAAATGGTGCTA
probe-miR-29c miR-29c ACCGATTTCAAATGGTGCTA probe-miR-301 miR-301
GCTTTGACAATACTATTGCACTG probe-miR-302a miR-302a
TCACCAAAACATGGAAGCACTTA probe-miR- miR-302a* AAAGCAAGTACATCCACGTTTA
302a* probe-miR-302b miR-302b CTACTAAAACATGGAAGCACTTA probe-miR-
miR-302b* AGAAAGCACTTCCATGTTAAAGT 302b* probe-miR-302c miR-302c
CCACTGAAACATGGAAGCACTTA probe-miR- miR-302c* CAGCAGGTACCCCCATGTTAAA
302c* probe-miR-302d miR-302d ACACTCAAACATGGAAGCACTTA
probe-miR-30a- miR-30a- GCTGCAAACATCCGACTGAAAG 3p 3p probe-miR-30a-
miR-30a- CTTCCAGTCGAGGATGTTTACA 5p 5p probe-miR-30b miR-30b
AGCTGAGTGTAGGATGTTTACA probe-miR-30c miR-30c
GCTGAGAGTGTAGGATGTTTACA probe-miR-30d miR-30d
CTTCCAGTCGGGGATGTTTACA probe-miR-30e- miR-30e-
GCTGTAAACATCCGACTGAAAG 3p 3p probe-miR-30e- miR-30e-
TCCAGTCAAGGATGTTTACA 5p 5p probe-miR-31 miR-31
CAGCTATGCCAGCATCTTGCC probe-miR-32 miR-32 GCAACTTAGTAATGTGCAATA
probe-miR-320 miR-320 TTCGCCCTCTCAACCCAGCTTTT probe-miR-323 miR-323
AGAGGTCGACCGTGTAATGTGC probe-miR-324- miR-324-
CCAGCAGCACCTGGGGCAGTGG 3p 3p probe-miR-324- miR-324-
ACACCAATGCCCTAGGGGATGCG 5p 5p probe-miR-325 miR-325
ACACTTACTGGACACCTACTAGG probe-miR-326 miR-326 CTGGAGGAAGGGCCCAGAGG
probe-miR-328 miR-328 ACGGAAGGGCAGAGAGGGCCAG probe-miR-329 miR-329
AAAGAGGTTAACCAGGTGTGTT probe-miR-33 miR-33 CAATGCAACTACAATGCAC
probe-miR-330 miR-330 TCTCTGCAGGCCGTGTGCTTTGC probe-miR-331 miR-331
TTCTAGGATAGGCCCAGGGGC probe-miR-335 miR-335 ACATTTTTCGTTATTGCTCTTGA
probe-miR-337 miR-337 AAAGGCATCATATAGGAGCTGGA probe-miR-338 miR-338
TCAACAAAATCACTGATGCTGGA probe-miR-339 miR-339 TGAGCTCCTGGAGGACAGGGA
probe-miR-33b miR-33b TGCAATGCAACAGCAATGCAC probe-miR-340 miR-340
GGCTATAAAGTAACTGAGACGGA probe-miR-342 miR-342
GACGGGTGCGATTTCTGTGTGAGA probe-miR-345 miR-345
GCCCTGGACTAGGAGTCAGCA probe-miR-346 miR-346 AGAGGCAGGCATGCGGGCAGACA
probe-miR-34a miR-34a AACAACCAGCTAAGACACTGCCA probe-miR-34b miR-34b
CAATCAGCTAATGACACTGCCTA probe-miR-34c miR-34c
GCAATCAGCTAACTACACTGCCT probe-miR-361 miR-361
GTACCCCTGGAGATTCTGATAA probe-miR-362 miR-362
CTCACACCTAGGTTCCAAGGATT probe-miR-363 miR-363
TTACAGATGGATACCGTGCAAT probe-miR-363* miR-363*
AAATTGCATCGTGATCCACCCG probe-miR-365 miR-365 ATAAGGATTTTTAGGGGCATTA
probe-miR-367 miR-367 TCACCATTGCTAAAGTGCAATT probe-miR-368 miR-368
AAACGTGGAATTTCCTCTATGT probe-miR-369- miR-369-
AAAGATCAACCATGTATTATT 3p 3p probe-miR-369- miR-369-
GCGAATATAACACGGTCGATCT 5p 5p probe-miR-370 miR-370
CCAGGTTCCACCCCAGCAGGC probe-miR-371 miR-371 ACACTCAAAAGATGGCGGCAC
probe-miR-372 miR-372 ACGCTCAAATGTCGCAGCACTTT probe-miR-373 miR-373
ACACCCCAAAATCGAAGCACTTC probe-miR-373* miR-373*
GGAAAGCGCCCCCATTTTGAGT probe-miR-374 miR-374 CACTTATCAGGTTGTATTATAA
probe-miR-375 miR-375 TCACGCGAGCCGAACGAACAAA probe-miR-376a
miR-376a ACGTGGATTTTCCTCTATGAT probe-miR- miR-376a*
CTCATAGAAGGAGAATCTACC 376a* probe-miR-376b miR-376b
AACATGGATTTTCCTCTATGAT probe-miR-377 miR-377 ACAAAAGTTGCCTTTGTGTGAT
probe-miR-378 miR-378 ACACAGGACCTGGAGTCAGGAG probe-miR-379 miR-379
TACGTTCCATAGTCTACCA probe-miR-380- miR-380- AAGATGTGGACCATATTACATA
3p 3p probe-miR-380- miR-380- GCGCATGTTCTATGGTCAACCA 5p 5p
probe-miR-381 miR-381 ACAGAGAGCTTGCCCTTGTATA probe-miR-382 miR-382
CGAATCCACCACGAACAACTTC probe-miR-383 miR-383 AGCCACAATCACCTTCTGATCT
probe-miR-384 miR-384 TATGAACAATTTCTAGGAAT probe-miR-409- miR-409-
AGGGGTTCACCGAGCAACATTCG 3p 3p probe-miR-409- miR-409-
TGCAAAGTTGCTCGGGTAACCT 5p 5p probe-miR-410 miR-410
AACAGGCCATCTGTGTTATATT probe-miR-411 miR-411 CGTACGCTATACGGTCTACTA
probe-miR-412 miR-412 ACGGCTAGTGGACCAGGTGAAGT probe-miR-421 miR-421
GCGCCCAATTAATGTCTGTTGAT probe-miR-422a miR-422a
GGCCTTCTGACCCTAAGTCCAG probe-miR-422b miR-422b
GGCCTTCTGACTCCAAGTCCAG probe-miR-423 miR-423 CTGAGGGGCCTCAGACCGAGCT
probe-miR-424 miR-424 TTCAAAACATGAATTGCTGCTG probe-miR-425 miR-425
GGCGGACACGACATTCCCGAT probe-miR-425- miR-425-
TCAACGGGAGTGATCGTGTCATT 5p 5p probe-miR-429 miR-429
ACGGTTTTACCAGACAGTATTA probe-miR-431 miR-431 TGCATGACGGCCTGCAAGACA
probe-miR-432 miR-432 CCACCCAATGACCTACTCCAAGA probe-miR-432*
miR-432* AGACATGGAGGAGCCATCCAG probe-miR-433 miR-433
ACACCGAGGAGCCCATCATGAT probe-miR-448 miR-448 ATGGGACATCCTACATATGCAA
probe-miR-449 miR-449 ACCAGCTAACAATACACTGCCA probe-miR-450 miR-450
TATTAGGAACACATCGCAAAAA probe-miR-451 miR-451
AAACTCAGTAATGGTAACGGTTT probe-miR-452 miR-452
GTCTCAGTTTCCTCTGCAAACA probe-miR-452* miR-452*
CTTCTTTGCAGATGAGACTGA probe-miR-453 miR-453
CGAACTCACCACGGACAACCTC
probe-miR-455 miR-455 CGATGTAGTCCAAAGGCACATA probe-miR-483 miR-483
AGAAGACGGGAGGAGAGGAGTGA probe-miR-484 miR-484
ATCGGGAGGGGACTGAGCCTGA probe-miR-485- miR-485- AGAGGAGAGCCGTGTATGAC
3p 3p probe-miR-485- miR-485- GAATTCATCACGGCCAGCCTCT 5p 5p
probe-miR-486 miR-486 CTCGGGGCAGCTCAGTACAGGA probe-miR-487a
miR-487a AACTGGATGTCCCTGTATGATT probe-miR-487b miR-487b
AAGTGGATGACCCTGTACGATT probe-miR-488 miR-488 TTGAGAGTGCCATTATCTGGG
probe-miR-489 miR-489 GCTGCCGTATATGTGATGTCACT probe-miR-490 miR-490
CAGCATGGAGTCCTCCAGGTTG probe-miR-491 miR-491
TCCTCATGGAAGGGTTCCCCACT probe-miR-492 miR-492
AAGAATCTTGTCCCGCAGGTCCT probe-miR-493 miR-493
AATGAAAGCCTACCATGTACAA probe-miR-493- miR-493-
CTGGCACACAGTAGACCTTCA 3p 3p probe-miR-494 miR-494
AAGAGGTTTCCCGTGTATGTTTCA probe-miR-495 miR-495
AAAGAAGTGCACCATGTTTGTTT probe-miR-496 miR-496 GAGATTGGCCATGTAAT
probe-miR-497 miR-497 ACAAACCACAGTGTGCTGCTG probe-miR-498 miR-498
GAAAAACGCCCCCTGGCTTGAAA probe-miR-499 miR-499
TTAAACATCACTGCAAGTCTTAA probe-miR-500 miR-500
CAGAATCCTTGCCCAGGTGCAT probe-miR-501 miR-501 TCTCACCCAGGGACAAAGGATT
probe-miR-502 miR-502 TAGCACCCAGATAGCAAGGAT probe-miR-503 miR-503
CTGCAGAACTGTTCCCGCTGCTA probe-miR-504 miR-504 ATAGAGTGCAGACCAGGGTCT
probe-miR-505 miR-505 GAGGAAACCAGCAAGTGTTGAC probe-miR-506 miR-506
TCTACTCAGAAGGGTGCCTTA probe-miR-507 miR-507 TTCACTCCAAAAGGTGCAAAA
probe-miR-508 miR-508 TCTACTCCAAAAGGCTACAATCA probe-miR-509 miR-509
TCTACCCACAGACGTACCAATCA probe-miR-510 miR-510
TGTGATTGCCACTCTCCTGAGTA probe-miR-511 miR-511 TGACTGCAGAGCAAAAGACAC
probe-miR-512- miR-512- GACCTCAGCTATGACAGCACTT 3p 3p probe-miR-512-
miR-512- GAAAGTGCCCTCAAGGCTGAGTG 5p 5p probe-miR-513 miR-513
ATAAATGACACCTCCCTGTGAA probe-miR-514 miR-514 CTACTCACAGAAGTGTCAAT
probe-miR-515- miR-515- ACGCTCCAAAAGAAGGCACTC 3p 3p probe-miR-515-
miR-515- CAGAAAGTGCTTTCTTTTGGAGAA 5p 5p probe-miR-516- miR-516-
ACCCTCTGAAAGGAAGCA 3p 3p probe-miR-516- miR-516-
AAAGTGCTTCTTACCTCCAGAT 5p 5p probe-miR-517* miR-517*
AGACAGTGCTTCCATCTAGAGG probe-miR-517a miR-517a
AACACTCTAAAGGGATGCACGAT probe-miR-517b miR-517b
AACACTCTAAAGGGATGCACGA probe-miR-517c miR-517c
ACACTCTAAAAGGATGCACGAT probe-miR-518a miR-518a
TCCAGCAAAGGGAAGCGCTTT probe-miR- miR-518a- AAAGGGCTTCCCTTTGCAGA
518a-2* 2* probe-miR-518b miR-518b ACCTCTAAAGGGGAGCGCTTTG
probe-miR-518c miR-518c CACTCTAAAGAGAAGCGCTTTG probe-miR- miR-518c*
CAGAAAGTGCTTCCCTCCAGAGA 518c* probe-miR-518d miR-518d
GCTCCAAAGGGAAGCGCTTTG probe-miR-518e miR-518e
ACACTCTGAAGGGAAGCGCTTT probe-miR-518f miR-518f
TCCTCTAAAGAGAAGCGCTTT probe-miR- miR-518f* AGAGAAAGTGCTTCCCTCTAGAG
518f* probe-miR-519a miR-519a GTAACACTCTAAAAGGATGCACTTT
probe-miR-519b miR-519b AAACCTCTAAAAGGATGCACTTT probe-miR-519c
miR-519c ATCCTCTAAAAAGATGCACTTT probe-miR-519d miR-519d
ACACTCTAAAGGGAGGCACTTTG probe-miR-519e miR-519e
ACACTCTAAAAGGAGGCACTTT probe-miR- miR-519e* GAAAGTGCTCCCTTTTGGAGAA
519e* probe-miR-520a miR-520a ACAGTCCAAAGGGAAGCACTTT probe-miR-
miR-520a* AGAAAGTACTTCCCTCTGGAG 520a* probe-miR-520b miR-520b
CCCTCTAAAAGGAAGCACTTT probe-miR-520c miR-520c
AACCCTCTAAAAGGAAGCACTTT probe-miR-520d miR-520d
AACCCACCAAAGAGAAGCACTTT probe-miR- miR-520d*
CAGAAAGGGCTTCCCTTTGTAGA 520d* probe-miR-520e miR-520e
CCCTCAAAAAGGAAGCACTTT probe-miR-520f miR-520f
AACCCTCTAAAAGGAAGCACTT probe-miR-520g miR-520g
ACACTCTAAAGGGAAGCACTTTGT probe-miR-520h miR-520h
ACTCTAAAGGGAAGCACTTTGT probe-miR-521 miR-521 ACACTCTAAAGGGAAGTGCGTT
probe-miR-522 miR-522 AACACTCTAAAGGGAACCATTTT probe-miR-523 miR-523
CCCTCTATAGGGAAGCGCGTT probe-miR-524 miR-524 ACTCCAAAGGGAAGCGCCTTC
probe-miR-524* miR-524* GAGAAAGTGCTTCCCTTTGTAG probe-miR-525
miR-525 AGAAAGTGCATCCCTCTGGAG probe-miR-525* miR-525*
GCTCTAAAGGGAAGCGCCTTC probe-miR-526a miR-526a AGAAAGTGCTTCCCTCTAGAG
probe-miR-526b miR-526b AACAGAAAGTGCTTCCCTCAAGAG probe-miR-
miR-526b* GCCTCTAAAAGGAAGCACTTT 526b* probe-miR-526c miR-526c
AACAGAAAGCGCTTCCCTCTAGAG probe-miR-527 miR-527
AGAAAGGGCTTCCCTTTGCAG probe-miR-532 miR-532 ACGGTCCTACACTCAAGGCATG
probe-miR-542- miR-542- TTTCAGTTATCAATCTGTCACA 3p 3p probe-miR-542-
miR-542- CTCGTGACATGATGATCCCCGA 5p 5p probe-miR-544 miR-544
ACTTGCTAAAAATGCAGAAT probe-miR-545 miR-545 CACACAATAAATGTTTGCTGAT
probe-miR-548a miR-548a GCAAAAGTAATTGCCAGTTTTG probe-miR-548b
miR-548b ACAAAAGCAACTGAGGTTCTTG probe-miR-548c miR-548c
GCAAAAGTAATTGAGATTTTTG probe-miR-548d miR-548d
GCAAAAGAAACTGTGGTTTTTG probe-miR-549 miR-549 AGAGCTCATCCATAGTTGTCA
probe-miR-550 miR-550 ATGTGCCTGAGGGAGTAAGACA probe-miR-551a
miR-551a TGGAAACCAAGAGTGGGTCGC probe-miR-552 miR-552
TTGTCTAACCAGTCACCTGTT probe-miR-553 miR-553 AAAACAAAATCTCACCGTTTT
probe-miR-554 miR-554 ACTGGCTGAGTCAGGACTAGC probe-miR-555 miR-555
ATCAGAGGTTCAGCTTACCCT probe-miR-556 miR-556 CATATTACAATGAGCTCATC
probe-miR-557 miR-557 AGACAAGGCCCACCCGTGCAAAC probe-miR-558 miR-558
ATTTTGGTACAGCAGCTCA probe-miR-559 miR-559 TTTTGGTGCATATTTACTTTA
probe-miR-560 miR-560 GGCGGCCGGCCGGCGCACGC probe-miR-561 miR-561
ACTTCAAGGATCTTAAACTTTG probe-miR-562 miR-562 GCAAATGGTACAGCTACTTT
probe-miR-563 miR-563 GGGAAACGTATGTCAACCT probe-miR-564 miR-564
GCCTGCTGACACCGTGCCT probe-miR-565 miR-565 AAACAGACATCGCGAGCCAGCC
probe-miR-566 miR-566 GTTGGGATCACAGGCGCCC probe-miR-567 miR-567
GTTCTGTCCTGGAAGAACATACT probe-miR-568 miR-568 GTGTGTATACATTTATACAT
probe-miR-569 miR-569 ACTTTCCAGGATTCATTAACT probe-miR-570 miR-570
TGCAAAGGTAATTGCTGTTTTC probe-miR-571 miR-571 CTCACTCAGATGGCCAACTCA
probe-miR-572 miR-572 TGGGCCACCGCCGAGCGGAC probe-miR-573 miR-573
CTGATCAGTTACACATCACTTCAG
probe-miR-574 miR-574 GTGGGTGTGTGCATGAGCGTG probe-miR-575 miR-575
GCTCCTGTCCAACTGGCTC probe-miR-576 miR-576 CAAAGACGTGGAGAAATTAGAAT
probe-miR-577 miR-577 CAGGTACCAATATTTTATCTA probe-miR-578 miR-578
ACAATCCTAGAGCACAAGAAG probe-miR-579 miR-579 ATCGCGGTTTATACCAAATGAAT
probe-miR-580 miR-580 CCTAATGATTCATCATTCTCAA probe-miR-581 miR-581
ACTGATCTAGAGAACACAAGA probe-miR-582 miR-582 AGTAACTGGTTGAACAACTGTAA
probe-miR-583 miR-583 GTAATGGGACCTTCCTCTTTG probe-miR-584 miR-584
CTCAGTCCCAGGCAAACCATAA probe-miR-585 miR-585 TAGCATACAGATACGCCCA
probe-miR-586 miR-586 GGACCTAAAAATACAATGCATA probe-miR-587 miR-587
GTGACTCATCACCTATGGAAA probe-miR-588 miR-588 GTTCTAACCCATTGTGGCCAA
probe-miR-589 miR-589 TCTGGGAACCGGCATTTGTTCTGA probe-miR-590
miR-590 CTGCACTTTTATGAATAAGCTC probe-miR-591 miR-591
ACAATGAGAACCCATGGTCT probe-miR-592 miR-592 ACATCATCGCATATTGACACAA
probe-miR-593 miR-593 GCTGAGCAATGCCTGGCTGGTGCCT probe-miR-594
miR-594 AAAGTCACAGGCCACCCCAGATGGG probe-miR-595 miR-595
AGACACACCACGGCACACTTC probe-miR-596 miR-596 CCCGAGGAGCCGGGCAGGCTT
probe-miR-597 miR-597 ACAGTGGTCATCGAGTGACACA probe-miR-598 miR-598
TGACGATGACAACGATGACGTA probe-miR-599 miR-599 GTTTGATAAACTGACACAAC
probe-miR-600 miR-600 GAGCAAGGCTCTTGTCTGTAAGT probe-miR-601 miR-601
CTCCTCCAACAATCCTAGACCA probe-miR-602 miR-602
GGGCCGCAGCTGTCGCCCGTGTC probe-miR-603 miR-603
GCAAAAGTAATTGCAGTGTGTG probe-miR-604 miR-604 GTCCTGAATTCCGCAGCCT
probe-miR-605 miR-605 AGGAGAAGGCACCATGGGATTTA probe-miR-606 miR-606
ATCTTTGATTTTCAGTAGTTT probe-miR-607 miR-607 GTTATAGATCTGGATTTGAAC
probe-miR-608 miR-608 ACGGAGCTGTCCCAACACCACCCCT probe-miR-609
miR-609 AGAGATGAGAGAAACACCCT probe-miR-610 miR-610
TCCCAGCACACATTTAGCTCA probe-miR-611 miR-611 GTCAGACCCCGAGGGGTCCTCGC
probe-miR-612 miR-612 AAGGAGCTCAGAAGCCCTGCCCAGC probe-miR-613
miR-613 GGCAAAGAAGGAACATTCCT probe-miR-614 miR-614
CCACCTGGCAAGAACAGGCGTTC probe-miR-615 miR-615 AGAGGGAGACCCAGGCTCGGA
probe-miR-616 miR-616 AAGTCACTGAAGGGTTTTGAGT probe-miR-617 miR-617
GCCACCTTCAAATGGGAAGTCT probe-miR-618 miR-618
ACTCAGAAGGACAAGTAGAGTTT probe-miR-619 miR-619
ACTGGGCACAAACATGTCCAGGTC probe-miR-620 miR-620 ATTTCTATATCTATCTCCAT
probe-miR-621 miR-621 AGGTAAGCGCTGTTGCTAGCC probe-miR-622 miR-622
GCTCCAACCTCAGCAGACTGT probe-miR-623 miR-623 ACCCAACAGCCCCTGCAAGGGAT
probe-miR-624 miR-624 TGAACACAAGGTACTGGTACTA probe-miR-625 miR-625
AGGACTATAGAACTTTCCCCCT probe-miR-626 miR-626 AAGACATTTTCAGACAGCT
probe-miR-627 miR-627 TCCTCTTTTCTTAGAGACTCAC probe-miR-628 miR-628
CGACTGCCACTCTTACTAGA probe-miR-629 miR-629 GCTGGGCTTACGTTGGGAGAAC
probe-miR-630 miR-630 ACCTTCCCTGGTACAGAATACT probe-miR-631 miR-631
GCTGAGGTCTGGGCCAGGTCT probe-miR-632 miR-632 TCCCACAGGAAGCAGACAC
probe-miR-633 miR-633 TTTATTGTGGTAGATACTATTAG probe-miR-634 miR-634
GTCCAAAGTTGGGGTGCTGGTT probe-miR-635 miR-635
GGACATTGTTTCAGTGCCCAAGT probe-miR-636 miR-636
CTGCGGGCGGGACGAGCAAGCACA probe-miR-637 miR-637
ACGCAGAGCCCGAAAGCCCCCAGT probe-miR-638 miR-638
AGGCCGCCACCCGCCCGCGATCCCT probe-miR-639 miR-639
ACAGCGCTCGCAACCGCAGCGAT probe-miR-640 miR-640 AGAGGCAGGTTCCTGGATCAT
probe-miR-641 miR-641 GAGGTGACTCTATCCTATGTCTTT probe-miR-642
miR-642 CAAGACACATTTGGAGAGGGAC probe-miR-643 miR-643
CTACCTGAGCTAGCATACAAGT probe-miR-644 miR-644 GCTCTAAGAAAGCCACACT
probe-miR-645 miR-645 TCAGCAGTACCAGCCTAGA probe-miR-646 miR-646
GCCTCAGAGGCAGCTGCTT probe-miR-647 miR-647 GAAGGAAGTGAGTGCAGCCAC
probe-miR-648 miR-648 ACCAGTGCCCTGCACACTT probe-miR-649 miR-649
GACTCTTGAACAACACAGGTTT probe-miR-650 miR-650 GTCCTGAGAGCGCTGCCTCCT
probe-miR-651 miR-651 CAAAAGTCAAGCTTATCCTAAA probe-miR-652 miR-652
TGCACAACCCTAGTGGCGCCATT probe-miR-653 miR-653 GTTCAGTAGAGATTGTTTCAA
probe-miR-654 miR-654 GCACATGTTCTGCGGCCCACCA probe-miR-655 miR-655
AAAGAGGTTAACCATGTATTAT probe-miR-656 miR-656 AGAGGTTGACTGTATAATATT
probe-miR-657 miR-657 CCTAGAGAGGGTGAGAACCTGCC probe-miR-658 miR-658
ACCAACGGACCTACTTCCCTCCGCC probe-miR-659 miR-659
TGGGGACCCTCCCTGAACCAAG probe-miR-660 miR-660 CAACTCCGATATGCAATGGGTA
probe-miR-661 miR-661 ACGCGCAGGCCAGAGACCCAGGCA probe-miR-662
miR-662 CTGCTGGGCCACAACGTGGGA probe-miR-663 miR-663
GCGGTCCCGCGGCGCCCCGCCT probe-miR-7 miR-7 CAACAAAATCACTAGTCTTCCA
probe-miR-9 miR-9 TCATACAGCTAGATAACCAAAGA probe-miR-9* miR-9*
ACTTTCGGTTATCTAGCTTTA probe-miR-92 miR-92 CAGGCCGGGACAAGTGCAATA
probe-miR-93 miR-93 CTACCTGCACGAACAGCACTTT probe-miR-95 miR-95
TGCTCAATAAATACCCGTTGAA probe-miR-96 miR-96 GCAAAAATGTGCTAGTGCCAAA
probe-miR-98 miR-98 AACAATACAACTTACTACCTCA probe-miR-99a miR-99a
CACAAGATCGGATCTACGGGTT probe-miR-99b miR-99b
CGCAAGGTCGGTTCTACGGGTG
[0023] Specifically, among the above-mentioned combinations,
methods, kits or biochips, the said evaluation of the physiological
and/or pathological condition of a subject is to determine the
physiological and/or pathological condition of the subject after
being administrated a test sample, which is specifically useful for
screening the test sample for the activities on the prevention
and/or treatment of diseases; the said evaluation of the
physiological and/or pathological condition of a subject is to
diagnose and/or differentially diagnose the diseases of the
subject; the said evaluation of the physiological and/or
pathological condition of a subject is to evaluate the
effectiveness of the treatment on the diseases of the subject; the
said evaluation of the physiological and/or pathological condition
of a subject is to predict the disease occurrence of the subject,
which is specifically the occurrence of complications and/or the
relapse of malignant diseases; the above-mentioned combinations,
methods, kits or biochips can also be useful for detecting the
subject for prohibited drugs-taking.
[0024] The above-mentioned diseases include a variety of tumors;
various acute/chronic infectious diseases, e.g. viral diseases such
as viral influenza, viral hepatitis, AIDS, SARS, bacterial diseases
such as tuberculosis, bacterial pneumonia, and other acute/chronic
infectious diseases caused by various pathogenic microorganisms;
other acute/chronic diseases such as diseases of respiratory
system, diseases of immune system, diseases of blood and
hematopoietic system, diseases of circulatory system such as
cardio-cerebrovascular diseases, metabolic diseases of endocrine
system, diseases of digestive system, diseases of nervous system,
diseases of urinary, diseases of reproductive system and diseases
of locomotor system.
[0025] The above-mentioned serum/plasma derives from the living
bodies, tissues, organs and/or corpuses of the subject.
[0026] The problems to be solved by the present invention include:
(1) analyzing and identifying the microRNA molecules and their
stability in serum/plasma of a variety of animals such as human,
mice and rats; (2) studying the specificity changes of microRNAs in
serum/plasma during the course of various clinical diseases
including a variety of tumors; various acute/chronic infectious
diseases, e.g. viral diseases such as viral influenza, viral
hepatitis, AIDS, SARS, bacterial diseases such as tuberculosis,
bacterial pneumonia, and other acute/chronic infectious diseases
caused by various pathogenic microorganisms; other acute/chronic
diseases such as diseases of respiratory system, diseases of immune
system, diseases of blood and hematopoietic system, diseases of
circulatory system such as cardio-cerebrovascular diseases,
metabolic diseases of endocrine system, diseases of digestive
system, diseases of nervous system, diseases of urinary system,
diseases of reproductive system and diseases of locomotor system;
(3) detecting the respective changes of microRNAs in serum/plasma
for different diseases through biochip and sequencing technology
for microRNAs in serum/plasma; (4) screening a kind of microRNA
molecules in serum/plasma which have relatively greater
differential expression during the course of diseases and normal
physiological conditions to develop detection technologies for
serum/plasma microRNAs, and then preparing biochips and diagnostic
kits useful for disease diagnosis etc.
[0027] Specifically, the present invention analyzes and identifies
the existence of microRNA molecules in serum/plasma of various
animals such as human, mice and rats through the methods of RT-PCR,
Real-time PCR, Northern blotting, RNase protection assay, Solexa
sequencing technology and biochip. The stability of microRNAs in
serum/plasma is studied by comparing the changes of microRNAs by
the effect of DNase and RNase. The existence of serum/plasma
microRNAs molecules and the correctness of their sequences are
further verified through sequencing and comparing the PCR products
of serum/plasma microRNAs.
[0028] The detailed preparation and analysis for serum/plasma
microRNAs are as follows:
[0029] RT-PCR method: collecting serum/plasma samples; conducting
reverse transcription reaction on serum/plasma samples to prepare
cDNA samples, or extracting total RNA of serum/plasma with Trizol
reagent and then conducting reverse transcription reaction so as to
prepare cDNA samples; designing a primer through mature microRNAs
so as to conduct PCR reaction; carrying out agarose gel
electrophoresis with the products of PCR; and observing and taking
photographs for the results under ultraviolet lamp after EB
staining.
[0030] Real-time PCR method: collecting serum/plasma samples;
conducting reverse transcription reaction on serum/plasma samples
to prepare cDNA samples, or extracting total RNA of serum/plasma
with Trizol reagent and then conducting reverse transcription
reaction so as to prepare cDNA samples; designing a primer of PCR
through mature microRNAs and adding a fluorescent probe EVA GREEN
so as to carry out PCR reaction; analyzing and processing the data
and then comparing the results.
[0031] Northern blotting method: collecting serum/plasma samples;
extracting total RNA of serum/plasma with Trizol reagent;
conducting denaturing PAGE-electrophoresis and membrane
transferring experiment; preparing isotope-labeled microRNA probes;
conducting membrane hybridization reaction; detecting the isotope
signal for results such as using phosphor-screen scanning
technology.
[0032] RNase protection assay: firstly synthesizing an antisense
RNA probe, labelling it with isotopes and purifying it; collecting
serum/plasma samples and extracting RNA; dissolving the extracted
DNA in a hybrid buffer and then adding an antisense RNA probe so as
to conduct hybridization reaction; adding a RNase digestion
solution to initate reaction; subjecting the resultant material to
electrophoresis and radioautography; and analyzing the results.
[0033] Solexa sequencing technology: collecting serum/plasma
samples; extracting total RNA of serum/plasma with Trizol reagent;
conducting PAGE-electrophoresis to recover RNA molecules of
17.about.27 nt; enzyme-linking adaptor prime to the 3' and 5' end
of small RNA molecules respectively; conducting RT-PCR reaction
prior to sequencing; and analyzing and processing the data.
[0034] Biochip method: arraying a library of all over 500 mature
microRNAs to prepare biochips; collecting serum/plasma samples;
extracting total RNA of serum/plasma; separating microRNAs by
column separation; fluorescently-labelling microRNAs by use of T4
RNA ligase; conducting hybridization reaction with a biochip; and
detecting and analyzing the data.
[0035] The change trend and change volume of serum/plasma microRNAs
during various diseases and their relevancy with various diseases
are analyzed through the above-mentioned technologies of RT-PCR,
Real-time PCR, Northern blotting, RNase protection assay, Solexa
sequencing technology, Biochip, etc. Among others, what to do
firstly is to detect and analyze the changes of let-7a, let-7b,
let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, miR-1, miR-100,
miR-101, miR-103, miR-105, miR-106a, miR-106b, miR-107, miR-10a,
miR-10b, miR-122a, miR-124a, miR-125a, miR-125b, miR-126, miR-126*,
miR-127, miR-128a, miR-128b, miR-129, miR-130a, miR-130b, miR-132,
miR-133a, miR-133b, miR-134, miR-135a, miR-135b, miR-136, miR-137,
miR-138, miR-139, miR-140, miR-141, miR-142-3p, miR-142-5p,
miR-143, miR-144, miR-145, miR-146a, miR-146b, miR-147, miR-148a,
miR-148b, miR-149, miR-150, miR-151, miR-152, miR-153, miR-154,
miR-154*, miR-155, miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p,
miR-181a, miR-181b, miR-181c, miR-181d, miR-182, miR-182*, miR-183,
miR-184, miR-185, miR-186, miR-187, miR-188, miR-189, miR-18a,
miR-18a*, miR-18b, miR-190, miR-191, miR-191*, miR-192, miR-193a,
miR-193b, miR-194, miR-195, miR-196a, miR-196b, miR-197, miR-198,
miR-199a, miR-199a*, miR-199b, miR-19a, miR-19b, miR-200a,
miR-200a*, miR-200b, miR-200c, miR-202, miR-202*, miR-203, miR-204,
miR-205, miR-206, miR-208, miR-20a, miR-20b, miR-21, miR-210,
miR-211, miR-212, miR-213, miR-214, miR-215, miR-216, miR-217,
miR-218, miR-219, miR-22, miR-220, miR-221, miR-222, miR-223,
miR-224, miR-23a, miR-23b, miR-24, miR-25, miR-26a, miR-26b,
miR-27a, miR-27b, miR-28, miR-296, miR-299-3p, miR-299-5p, miR-29a,
miR-29b, miR-29c, miR-301, miR-302a, miR-302a*, miR-302b,
miR-302b*, miR-302c, miR-302c*, miR-302d, miR-30a-3p, miR-30a-5p,
miR-30b, miR-30c, miR-30d, miR-30e-3p, miR-30e-5p, miR-31, miR-32,
miR-320, miR-323, miR-324-3p, miR-324-5p, miR-325, miR-326,
miR-328, miR-329, miR-33, miR-330, miR-331, miR-335, miR-337,
miR-338, miR-339, miR-33b, miR-340, miR-342, miR-345, miR-346,
miR-34a, miR-34b, miR-34c, miR-361, miR-362, miR-363, miR-363*,
miR-365, miR-367, miR-368, miR-369-3p, miR-369-5p, miR-370,
miR-371, miR-372, miR-373, miR-373*, miR-374, miR-375, miR-376a,
miR-376a*, miR-376b, miR-377, miR-378, miR-379, miR-380-3p,
miR-380-5p, miR-381, miR-382, miR-383, miR-384, miR-409-3p,
miR-409-5p, miR-410, miR-411, miR-412, miR-421, miR-422a, miR-422b,
miR-423, miR-424, miR-425, miR-425-5p, miR-429, miR-431, miR-432
miR-432*, miR-433, miR-448, miR-449, miR-450, miR-451, miR-452,
miR-452*, miR-453, miR-455, miR-483, miR-484, miR-485-3p,
miR-485-5p, miR-486, miR-487a, miR-487b, miR-488, miR-489, miR-490,
miR-491, miR-492, miR-493, miR-493-3p, miR-494, miR-495, miR-496,
miR-497, miR-498, miR-499, miR-500, miR-501, miR-502, miR-503,
miR-504, miR-505, miR-506, miR-507, miR-508, miR-509, miR-510,
miR-511, miR-512-3p, miR-512-5p, miR-513, miR-514, miR-515-3p,
miR-515-5p, miR-516-3p, miR-516-5p, miR-517*, miR-517a, miR-517b,
miR-517c, miR-518a, miR-518a-2*, miR-518b, miR-518c, miR-518c*,
miR-518d, miR-518e, miR-518f, miR-518f*, miR-519a, miR-519b,
miR-519c, miR-519d, miR-519e, miR-519e*, miR-520a, miR-520a*,
miR-520b, miR-520c miR-520d, miR-520d*, miR-520e, miR-520f,
miR-520g, miR-520h, miR-521, miR-522, miR-523, miR-524, miR-524*,
miR-525, miR-525*, miR-526a, miR-526b, miR-526b*, miR-526c,
miR-527, miR-532, miR-542-3p, miR-542-5p, miR-544, miR-545,
miR-548a, miR-548b, miR-548c, miR-548d, miR-549, miR-550, miR-551a,
miR-552, miR-553, miR-554, miR-555, miR-556, miR-557, miR-558,
miR-559, miR-560, miR-561, miR-562, miR-563, miR-564, miR-565,
miR-566, miR-567, miR-568, miR-569, miR-570, miR-571, miR-572,
miR-573, miR-574, miR-575, miR-576, miR-577, miR-578, miR-579,
miR-580, miR-581, miR-582, miR-583, miR-584, miR-585, miR-586,
miR-587, miR-588, miR-589, miR-590, miR-591, miR-592, miR-593,
miR-594, miR-595, miR-596, miR-597, miR-598, miR-599, miR-600,
miR-601, miR-602, miR-603, miR-604, miR-605, miR-606, miR-607,
miR-608, miR-609, miR-610, miR-611 miR-612, miR-613, miR-614,
miR-615, miR-616, miR-617, miR-618, miR-619, miR-620, miR-621,
miR-622, miR-623, miR-624, miR-625, miR-626, miR-627, miR-628,
miR-629, miR-630, miR-631, miR-632, miR-633, miR-634, miR-635,
miR-636, miR-637, miR-638, miR-639, miR-640, miR-641, miR-642,
miR-643, miR-644, miR-645, miR-646, miR-647, miR-648, miR-649,
miR-650, miR-651, miR-652, miR-653, miR-654, miR-655, miR-656,
miR-657, miR-658, miR-659, miR-660, miR-661, miR-662, miR-663,
miR-7, miR-9, miR-9*, miR-92, miR-93, miR-95, miR-98, miR-99a and
miR-99b in various clinical diseases (including a variety of
tumors; various acute/chronic infectious diseases, e.g. viral
diseases such as viral influenza, viral hepatitis, AIDS, SARS,
bacterial diseases such as tuberculosis, bacterial pneumonia, and
other acute/chronic infectious diseases caused by various
pathogenic microorganisms; other acute/chronic diseases such as
diseases of respiratory system, diseases of immune system, diseases
of blood and hematopoietic system, diseases of circulatory system
such as cardio-cerebrovascular diseases, metabolic diseases of
endocrine system, diseases of digestive system, diseases of nervous
system, diseases of urinary system, diseases of reproductive system
and diseases of locomotor system); Biochips of serum/plasma
microRNAs are prepared to determine the changes of serum/plasma
microRNAs in different diseases, and meanwhile, Solexa sequencing
and analysis on microRNAs in serum/plasma in different diseases are
conducted.
[0036] The research and development of a technology for detecting
disease-related serum/plasma microRNAs. Specifically, the microRNAs
with disease-related specificity changes are screened out, their
primers are collected into a PCR kit (RT-PCR or Real-time PCR) to
prepare a disease-diagnostic kit, or their reverse complementary
sequences are dripped on chips as probes so as to prepare the
biochips for detecting serum/plasma microRNAs specific for a
certain disease.
[0037] Presently, the technologies of traditional biochemistry and
molecular biology for the clinical diagnosis of diseases are
relatively complicated and insensitive. Novel techniques developed
in recent years possibly useful for disease diagnosis are gene chip
technique, protein (antibody) chip technique, etc. The changes at
mRNA level measured through gene chips cannot completely reflect
the actual changes at protein level, since the bioactivity of
protein is closely related to post-transcriptional modification
such as glycosylation and phosphorylation. In addition, for
detection of many diseases, marker molecules in body fluids and
blood cannot be detected through gene chip technology. Meanwhile,
protein (antibody) chip technique and proteomic techniques also
bear their limitations. In human body, especially in serum/plasma,
there are tens of thousands of protein and polypeptide segments
with extensively distributed concentrations, and the number of
proteins definitely reported is very small, let alone those
quantified. It is an extremely arduous task to find out those
proteins having close relation with specific diseases from the
large quantity of proteins and understand their roles in
histopathologic changes. Moreover, lacking of complete antibody
resources is the bottleneck restraining the development of antibody
biochip technology. The detection technology for serum/plasma
microRNAs based on biochips of serum/plasma microRNAs and
diagnostic kits skillfully combines the peculiar properties of
serum/plasma microRNAs with conventional molecular biology
detection technique together, which can rapidly analyze the
respective constitution of serum/plasma microRNAs in respect of
various diseases with high throughput and hence be of extremely
clinical practicality. Since the changes of physiological
conditions in organs and tissues will cause the constitutional
changes of serum/plasma microRNAs, serum/plasma microRNAs can be
used as "fingerprints for diseases" to realize early diagnosis of
diseases.
[0038] The advantages of the technology of detecting serum/plasma
microRNAs are as follows:
[0039] (1) As novel disease markers, serum/plasma microRNAs possess
certain advantages such as extensive spectrum for detection, high
sensitivity, low cost for detection, convenient sampling, easy
preservation for samples (preserving serum/plasma at -20.degree. C.
will do), etc. This method can be widely used in general survey of
diseases and other relevant tasks and has become an efficient means
for early diagnosis of diseases.
[0040] (2) As novel disease markers, serum/plasma microRNAs will
improve the low-specificity and low-sensitivity caused by
individual differences which single markers are difficult to
overcome, and notably increase the clinical detection rate of
diseases so as to realize early diagnosis of diseases.
[0041] (3) The advantages of the technology of detecting
serum/plasma microRNAs lie in that what to be detected is series of
disease related markers, thus it can address the differences (i.e.,
age, sex, race, diet, circumstance, etc.) between individual
patients, which are exactly a primary problem difficult to overcome
by single disease markers.
[0042] In summary, utilizing the technology of detecting
serum/plasma microRNAs can confirm diagnosis of histopathologic
changes in early stage. These novel serum/plasma markers not only
provide material foundation for people to comprehensively
understand the mechanism of histopathologic changes in molecule
level, but also accelerate the progress in diagnostics and
therapeutics of clinical diseases. Of course, a majority of
molecular diagnostic techniques used for disease detection in early
period are at initial experimental stage and their validity needs
to be further verified and improved. Moreover, since every disease
has the characteristics of its own, this requires a peculiar method
for the detection of said disease. In this manner, it is impossible
for all diseases to be detected out only through one or only a few
of detection methods. Nevertheless, based on the superiority of
serum/plasma microRNAs, it is believed that, in the near future,
the diagnostic technique of serum/plasma microRNAs for severe
diseases such as cancer will become part of routine physical
examination. In addition, microRNA related gene therapy will be
widely utilized. Consequently, the overcoming of these diseases
will come true, not just a dream.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] The following are the detailed description of the
embodiments of this invention with reference to the drawings,
wherein:
[0044] FIG. 1 shows the RT-PCR result of partial microRNAs directly
detected in the serum of a normal person.
[0045] FIG. 2 shows the RT-PCR results of the microRNAs in the RNA
extracted from the serum of a normal person.
[0046] In FIG. 1 and FIG. 2, U6 is a snRNA with a molecular weight
of 100 bp, serving as an internal reference molecule in microRNAs
experiments. The rest of 12 microRNAs are each miR-181a(181a),
miR-181b(181b), miR-223(223), miR-142-3p(142-3p),
miR-142-5p(142-5p), miR-150(150) with blood cell specificity;
miR-1(1), miR-133a(133a), miR-206(206) from cardiac muscles and
skeletal muscles; miR-9(9), miR-124a(124a) from brain tissues; and
miR-122a (122a) from liver.
[0047] FIG. 3 shows the RT-PCR results of partial micro-RNAs
directly detected in the serum of mouse, rat, fetal bovine, calf
and horse respectively.
[0048] FIG. 4 shows the variable quantity of the partial microRNAs
in the serum of a patient suffering from the shown diseases
compared with microRNAs in the serum of a normal person.
[0049] FIG. 5 shows the ratio between the quantities of macroRNAs
and microRNAs in blood cells and serum.
[0050] FIG. 6 shows the enzyme digested results of macroRNAs and
microRNAs.
THE BEST MODE FOR CARRYING OUT THE INVENTION
Example 1
[0051] The RT-PCR Experiments of MicroRNAs in Serum/Plasma
[0052] By using RT-PCR technique, it is found and proved that there
stably exist various microRNAs in serum/plasma of both human beings
and animals, and that their expression levels are considerably
high. The specific RT-PCR steps are as follows:
[0053] (1) collecting serum/plasma of mice, rats, normal persons
and some patients;
[0054] (2) preparing samples of cDNA. This operation has two
options: one is to directly conduct reverse transcription reaction
using 10 .mu.l of serum/plasma; the other is to firstly extract the
total RNA from serum/plasma (usually, about 10 .mu.g of RNA can be
enriched from 10 ml of serum/plasma) with Trizol reagent
(Invitrogen Co.), subsequently obtain cDNA through RNA reverse
transcription reaction. The reaction system of reverse
transcription includes 4 .mu.l 5.times.AMV buffer, 2 .mu.l 10 mM
each dNTP mixture (Takara Co.), 0.5 .mu.l RNase Inhibitor (Takara
Co.), 2 .mu.l AMY (Takara Co.) and 1.5 .mu.l gene specific reverse
primers mixtures. The reaction steps successively include 15
minutes of incubation at 16.degree. C., 1 hour of reaction at
42.degree. C. and 5 minutes of incubation at 85.degree. C.;
[0055] (3) PCR and Electrophoresis observation. The cDNA is diluted
by 1/50. To 1 .mu.l diluted cDNA are added 0.3 .mu.l Taq polymerase
(Takara Co.), 0.2 .mu.l 10 .mu.M forward primer, 0.20 .mu.l 10
.mu.M universal reverse primer, 1.2 .mu.l 25 mM MgCl.sub.2, 1.6
.mu.l 2.5 mM each dNTP mixture (Takara Co.), 2 .mu.l 10.times.PCR
buffer, 13.5 .mu.l H.sub.2O, and PCR reaction is conducted in the
20 .mu.l system. The PCR reaction is done under the following
conditions: one cycle at 95.degree. C. for 5 mins followed by 40
cycles at 95.degree. C. for 15 seconds and 60.degree. C. for 1
minute. 10 .mu.l PCR product is subjected to 3% Agarose Gel
Electrophoresis, which is observed under ultraviolet lamp after EB
staining.
[0056] The detailed experimental results are shown in FIG. 1. FIG.
1 shows the experimental results of RT-PCR directly conducted on
the serum of normal persons. The all over 500 mature microRNAs in
human being are selected for conducting RT-PCR reaction, of which
12 microRNAs are shown in FIG. 1 and each miR-181a, miR-181b,
miR-223, miR-142-3p, miR-142-5p, miR-150 with is blood cell
specificity; miR-1, miR-133a, miR-206 from cardiac muscles and
skeletal muscles; miR-9 and miR-124a from brain tissues; and
miR-122a from liver. It can be seen from the results that all
microRNAs from the above-mentioned four tissues are detectable in
blood, and that not all over 500 mature microRNAs have high
expression level in the serum/plasma, with some microRNAs being in
fairly trace amount and even being normally nondetectable.
[0057] To further verify that there stably exist the microRNAs in
serum/plasma, RNA is firstly extracted from the serum of normal
persons, then all over 500 mature microRNAs of human are selected
for PCR experiment. As shown in FIG. 2, the results of FIG. 2 is
quite consistent with that of FIG. 1, the singleness of the PCR
products indicating that both two assays can detect the expression
and level of the microRNAs in people's serum/plasma, and proving
that there stably exist microRNAs of various tissues sources in
people's serum/plasma. In addition, the same method is used to
detect the expression and level of over 500 microRNAs in the
serum/plasma of mouse, rat, fetal bovine, calf and horse, it is
also found that there is stable expression of microRNAs of various
tissues sources in serum/plasma of mouse, rat, fetal bovine, calf
and horse (see FIG. 3).
Example 2
The Real-Time PCR Experiments of MicroRNAs in Serum/Plasma
[0058] Quantitative PCR experiments of microRNAs in serum/plasma
are conducted to study the specific variation of microRNAs quantity
in serum/plasma during the course of various diseases, including
various tumors, various acute and chronic infectious diseases, e.g.
viral diseases such as viral influenza, viral hepatitis, AIDS,
SARS, bacterial diseases such as tuberculosis, bacterial pneumonia,
and other acute and chronic infectious diseases caused by various
pathogenic microorganisms; other acute and chronic diseases such as
diseases of respiratory system, diseases of immune system, diseases
of blood and hematopoietic system, diseases of circulatory system
such as cardio-cerebrovascular disease, metabolic diseases of
endocrine system, diseases of digestive system, diseases of nervous
system, diseases of urinary system, diseases of reproductive system
and diseases of locomotor system. The experimental principles and
experimental steps of quantitative PCR are basically the same as
those of RT-PCR, with the only difference between them being the
addition of a fluorescent dye EVA GREEN in the process of PCR. An
ABI Prism 7300 fluorescent quantitative PCR instrument is used to
conduct PCR reaction under the following conditions: one cycle at
95.degree. C. for 5 mins followed by 40 cycles at 95.degree. C. for
15 seconds and 60.degree. C. for 1 minute. The data processing
method used is .DELTA..DELTA.CT method, wherein CT is the number of
cycles when the reaction reaches the threshold. The expression
level of each microRNAs relative to that of internal standard
reference can be expressed by the equation of 2-ACT, wherein
.DELTA.CT=CT.sub.sample-CT.sub.internal reference. Reverse
transcription reactions are directly conducted on serum/plasma
samples of a patient and those of a normal person, and the
quantities of microRNAs contained in each sample of serum/plasma
are compared through quantitative PCR reactions.
[0059] Serum samples of patients who suffer from aplastic anemia,
breast cancer, osteosarcoma, CNS (Central Nervous System) lymphoma,
diabetes are selected, and at the same time, all over 500 mature
microRNAs of human beings are used to conduct PCR reaction
experiments. FIG. 4 shows the quantitative PCR experimental results
of microRNAs within serum of patients and normal persons which
include the above-mentioned miR-181a, miR-181b, miR-223,
miR-142-3p, miR-142-5p, miR-150 with blood cell specificity; miR-1,
miR-133a, miR-206 from cardiac muscles and skeletal muscles; miR-9,
miR-124a from brain tissues; and miR-122a from liver. The ratio of
the microRNAs quantity in serum between normal persons and patients
suffering from aplastic anemia, breast cancer, osteosarcoma, CNS
(Central Nervous System) lymphoma, diabetes are respectively
up-regulated or down-regulated, and the variation extent of the
microRNAs quantity from the same tissue source differs in patients
with different diseases, indicating that there is specificity
variation of microRNAs quantity in the serum/plasma of patients
with different diseases. They can be taken as a type of novel
markers for disease diagnosis.
Example 3
The Superiority of Serum/Plasma MicroRNAs as Disease Markers
[0060] Through detecting the quantities of microRNAs and macroRNAs
in serum and blood cells, it is found that there is an abundant
content of microRNAs in serum. See FIG. 5. As represented by U6
molecules with a molecular weight of 100 bp and ribosomal RNA
molecules with molecular weights being 18S and 28S respectively,
the quantity of macroRNAs in blood cells is at least tens times
that in serum; while the quantity of microRNAs in serum remains the
same as that in blood cells except the microRNAs with blood cell
specificity. Therefore, serum/plasma will specifically enrich small
molecule RNAs, especially microRNAs.
[0061] It is also found that microRNAs are to some extent able to
resist the action of endonuclease, which is possibly one of the
reasons why microRNAs can stably exist in serum/plasma. Total RNAs
extracted from cultured cell line are processed with endonuclease
RNase A and the remaining quantity of macroRNAs and microRNAs are
then detected. As shown in FIG. 6, it is found that microRNAs can
to some extent resist the degradation of endonuclease while the
macroRNAs are substantially completely cut off. Therefore microRNAs
can stably exist in serum/plasma.
[0062] Based on the two characteristics of abundance in content and
stable existence of microRNAs in serum/plasma, microRNAs could be
well applied in clinical test.
Example 4
Preparation of the Biochip of Serum/Plasma MicroRNAs Useful for
Disease Diagnosis
[0063] A biochip of serum/plasma microRNAs is fabricated to verify
the reliability of a kind of serum/plasma microRNAs probes relating
to diseases which are selected through quantitative PCR method. The
biochip contains all microRNAs probes that can be normally detected
in people's serum/plasma, constituting a probe library. See Table
1.
[0064] When the probes are specifically applied in certain disease
diagnosis or efficacy screening, some probes of the probe library
are put together to construct a probe collection which makes it
possible to quantitatively detect the variation of microRNAs in the
specific conditions. For example, when diagnosing colon cancer, the
collection of probes that have interaction with microRNAs of
numbers 17-5p, 21, 103, 106a, 107, 126*, 143, 145, 150, 155 and 210
is used. For another example, when diagnosing myocardial
hypertrophy and chronic heart failure, the collection of probes
that have interaction with microRNAs of numbers 21, 23a, 23b, 24,
27a, 27b, 125b, 195, 199a, 214, 217, 133a is used. In addition, the
chip can also do high-throughput screening of the probes of
microRNAs varying stably in serum/plasma, and diseases can be
predicted and diagnosed based on the overall variation of microRNAs
in serum/plasma.
[0065] Sequencing method or quantitative PCR method is firstly used
to determine that there is more than one copy of microRNAs in
serum/plasma, and then reverse complementary probes of these
microRNAs are synthesized, after which these probes are spotted on
a chemically-modified slide in a size of 75.times.25 mm using a
biochip microarrayer SmartArray.TM.. The samples spotted on the
chip also include U6 and tRNA as internal standard,
artificially-prepared external standard in length of 30 bases, Hex
as positive control etc. The entire lattice is divided into 4
sub-lattices and each sub-lattice has 23 rows and 21 columns,
wherein the spot distance is 185 mm and the spot diameter is about
130 .mu.m and each probe was repeatly spotted for 3 times.
[0066] The operational procedure of the biochip is: (1) extracting
the total RNA from serum/plasma and detecting its quality through
formaldehyde denaturing gel electrophoresis; (2) separation of
microRNAs: 50-100 .mu.g total RNA is taken to separate microRNAs
from total RNA with Ambion's miRNA Isolation Kit (Cat #. 1560); (3)
fluorescently-labeling of microRNAs samples: microRNAs samples are
fluorescently-labeling with T4 RNA ligase, then precipitated with
absolute ethanol, and then blown to dryness for chip hybridization;
(4) hybridization and cleaning: RNA is dissolved into 16 .mu.L
hybridizing solution (15% formamide, 0.2% SDS, 3.times.SSC and
50.times.Denhardt's solution), and hybridized at 420 overnight.
After completion of the hybridization, it is washed in a solution
containing 0.2% SDS and 2.times.SSC at about 42.degree. C. for 4
minutes, and then washed in a solution containing 0.2.times.SSC at
room temperature for 4 minutes. Thereafter, the slides can be used
for scanning immediately after being dried; (5) chip scanning: the
chip is scanned with two-channel laser scanner LuxScan 10K/A; (6)
data extracting and analysis: the chip image is analyzed with an
image analyzing software LuxScan 3.0, the image signal is
transformed into digital signal, and finally
differentially-expressed genes are analyzed and selected with SAM
method.
[0067] A biochip is prepared as above by using a kind of
serum/plasma microRNAs probes which express greatly differently
under disease condition and normal physiological condition
double-verified by quantitative PCR technique and biochip
technique. As compared with the traditional chip, there is no
significant improvement in the manufacturing process and
operational procedure of this biochip, but this chip simplifies the
probe library, thereby greatly reducing the manufacturing cost and
production time of the chip, and hence is easy to prepare.
Meanwhile it increases the pertinence and practicability of chip.
The application of the chip in practice can detect diseases in an
early phase with only need of the serum/plasma of a patient and no
need of other tissues, which helps guide the diagnosis and
treatment.
Example 5
Preparation of Kits of MicroRNAs Useful for Disease Diagnosis and
Prediction
[0068] The manufacturing processed and operational procedures of
microRNAs kits useful for diagnosis, prediction of complication
occurrence and malignant disease relapse, evaluation of therapeutic
effects, screening of pharmaceutical active ingredients, assessment
of drug efficacy, forensic authentication and prohibited drug
inspection, etc. of all diseases are based on quantitative PCR
technique and semi-quantitative PCR technique and biochip
technique. The above-mentioned diseases include various tumors;
various acute/chronic infectious diseases, e.g. viral diseases such
as viral influenza, viral hepatitis, AIDS, SARS, bacterial diseases
such as tuberculosis, bacterial pneumonia, and other acute/chronic
infectious diseases caused by various pathogenic microorganisms;
other acute/chronic diseases such as diseases of respiratory
system, diseases of immune system, diseases of blood and
hematopoietic system, diseases of circulatory system such as
cardio-cerebrovascular diseases, metabolic diseases of endocrine
system, diseases of digestive system, diseases of nervous system,
diseases of urinary system, diseases of reproductive system and
diseases of locomotor system.
[0069] Sequencing method or quantitative PCR method is firstly used
to determine that there is more than one copy of microRNAs in
serum/plasma. Then, a kind of serum/plasma microRNAs that have a
big difference between the expression levels in disease condition
and in normal physiological condition are screened out through the
techniques of quantative PCR and biochip, which are taken as an
indicator for predicting whether canceration or other disease
occurs and diagnosing the pathological degree. Finally the number
of screened corresponding serum/plasma microRNAs of each disease
would be controlled to over ten to tens, which is the optimized
condensement of the chip-probe library. The kit contains a batch of
serum/plasma microRNAs primers, Taq polymerase, dNTP, etc. The
value of the kit lies in making it possible to detect the changing
trend of microRNAs through the most simplified probe library and
with only need of serum/plasma and no need of any other tissue
samples, and further predict the probability of occurrence of
diseases or diagnose the pathological phase of diseases based on
this changing trend detected. Thus, the application of this kit in
practice can increase the possibility of discovering diseases in an
early phase, which helps guide the diagnosis and treatment of
diseases.
* * * * *