U.S. patent application number 12/444447 was filed with the patent office on 2010-04-22 for stabilizing compositions and methods for extraction of ribonucleic acid.
This patent application is currently assigned to DNA GENOTEK INC.. Invention is credited to Hyman Chaim Birnboim, Adele Jackson.
Application Number | 20100099149 12/444447 |
Document ID | / |
Family ID | 39268089 |
Filed Date | 2010-04-22 |
United States Patent
Application |
20100099149 |
Kind Code |
A1 |
Birnboim; Hyman Chaim ; et
al. |
April 22, 2010 |
STABILIZING COMPOSITIONS AND METHODS FOR EXTRACTION OF RIBONUCLEIC
ACID
Abstract
The present invention provides a composition and method for
stabilizing ribonucleic acid (RNA) from biological samples such
that the ribonucleic acid within the sample remains stable at room
temperature. The composition comprises an anionic detergent and a
buffering agent at a pH of about 5 to about 8.2 and is used in
methods for extracting and storing ribonucleic acid from the
biological sample.
Inventors: |
Birnboim; Hyman Chaim;
(Ottawa, CA) ; Jackson; Adele; (Stittsville,
CA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP;FLOOR 30, SUITE 3000
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Assignee: |
DNA GENOTEK INC.
Ottawa
ON
|
Family ID: |
39268089 |
Appl. No.: |
12/444447 |
Filed: |
October 5, 2007 |
PCT Filed: |
October 5, 2007 |
PCT NO: |
PCT/CA2007/001785 |
371 Date: |
October 22, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60828563 |
Oct 6, 2006 |
|
|
|
60866985 |
Nov 22, 2006 |
|
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60949778 |
Jul 13, 2007 |
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Current U.S.
Class: |
435/91.3 ;
252/380; 536/23.1 |
Current CPC
Class: |
C12N 15/1003 20130101;
C12Q 1/6806 20130101; C12Q 1/6806 20130101; C12Q 2527/125
20130101 |
Class at
Publication: |
435/91.3 ;
252/380; 536/23.1 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C09K 3/00 20060101 C09K003/00; C07H 21/02 20060101
C07H021/02 |
Claims
1. A composition for extracting and storing ribonucleic acid from a
sample such that the ribonucleic acid within said sample remains
stable at room temperature, said composition comprises: a. an
anionic detergent; and b. a buffering agent at a pH of about 5 to
about 8.2; wherein when said composition is mixed with said sample
to form a liquid mixture, the ribonucleic acid in said liquid
mixture is stable at room temperature.
2. The composition of claim 1, wherein said anionic detergent is
sodium dodecyl sulphate, sodium lauroyl sarcosinate (Sarkosyl),
lithium dodecyl sulphate or sodium 1-octane sulfonic acid.
3. The composition of claim 1 or 2, wherein said anionic detergent
is sodium dodecyl sulphate or sodium lauroyl sarcosinate.
4. The composition of claim 3, wherein the sodium dodecyl sulphate
or sodium lauroyl sarcosinate are at a concentration of from about
0.5% to about 8% when mixed with said sample.
5. The composition of any one of claims 1 to 4, wherein said
buffering agent is at a pH of about 5.1 to about 7, about 5.5 to
about 7.5, about 6.5 to about 7.0 or about 6.8.
6. The composition of any one of claims 1 to 5, wherein said
buffering agent is sodium cyclohexane diaminetetraacetate (CDTA),
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), acetic
acid or acetate (e.g. sodium acetate), citric acid or citrate,
malic acid, phthalic acid, succinic acid, histidine, pyrophosphoric
acid, maleic acid, cacodylic acid, .beta..beta.'-Dimethylglutaric
acid, carbonic acid or carbonate, 5(4)-Hydroxymethylimidazole,
glycerol 2-phosphoric acid, ethylenediamine, imidazole, arsenic
acid, phosphoric acid or phosphate, sodium acetate, 2:4:6
collidine, 5(4)-methylimidazole, N-ethylmorpholine,
triethanolamine, diethylbarbituric acid,
tris(hydroxymethyl)aminomethane (Tris),
3-(N-Morpholino)propanesulfonic acid; 4-morpholinepropanesulfonic
acid (MOPS), 2-morpholinoethanesulfonic acid (MES),
piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES),
N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES),
4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), analogues
thereof, or combinations thereof.
7. The composition of any one of claims 1 to 5, wherein said buffer
is a phosphate buffer, a carbonate buffer, an ethylenediamine
buffer or an imidazole buffer.
8. The composition of any one of claims 1 to 6, wherein the
buffering agent is CDTA or citric acid.
9. The composition of claim 1, wherein said sample is a bodily
fluid or a bodily tissue from a mammal.
10. The composition of claim 9, wherein said mammal is a human or a
cow.
11. The composition of claim 1, wherein said sample is from a
human; a non-human primate; livestock including cattle, pigs,
sheep, goats or domestic birds including chicken, turkey, pheasant,
duck or geese); game or wild animals including deer, elk, moose,
fish, birds or bears; laboratory or companion animals including
non-human primates, rodents including mice, rats, rabbits, guinea
pigs, gerbils or hamsters; dogs; cats; fish; snakes; lizards;
turtles; a horse; plants; plant parts; cell lines; soil
microorganisms; sewage microorganisms; or pathogenic microorganisms
including virus, bacteria or parasites.
12. The composition of any one of claims 1 to 11, wherein the
composition stabilized said ribonucleic acid at room temperature
for at least about one day, two days, three days, four days, five
days, six days, one week, two weeks, three weeks, four weeks, five
weeks, six weeks, seven weeks or eight weeks, about one day to
about eight weeks, or greater than about eight weeks.
13. The composition of any one of claims 1 to 12, wherein the
composition stabilizes said ribonucleic acid at room temperature
for about one day to about eight weeks at room temperature.
14. A method for preserving ribonucleic acid from a biological
sample comprising the steps of: a. obtaining the sample from a
subject; b. contacting said sample with a composition comprising an
anionic detergent and a buffering agent at a pH of about 5 to about
8.2 to form a liquid mixture; c. storing the mixture at room
temperature; and d. heating the mixture at greater than or about
equal to 50.degree. C. prior to subsequent processing, wherein said
composition stabilizes said ribonucleic acid at room
temperature.
15. The method of claim 14, wherein step d further comprises
contacting the mixture with a protease.
16. The method of claim 15, wherein the protease is proteinase
K.
17. The method of any one of claims 14 to 16 further comprising the
step of (e) heating the mixture at greater than or equal to about
90.degree. C. prior to subsequent processing and before or after
heating step d.
18. The method of anyone of claims 14 to 17, wherein said anionic
detergent is sodium dodecyl sulphate, sodium lauroyl sarcosinate,
lithium dodecyl sulphate or sodium 1-octane sulfonic acid.
19. The method of any one of claims 14 to 18, wherein said anionic
detergent is sodium dodecyl sulphate or sodium lauroyl
sarcosinate.
20. The method of claim 19, wherein the sodium dodecyl sulphate or
sarkoryl are at a concentration of from about 0.5% to about 8%.
21. The method of any one of claims 14 to 20, wherein said
buffering agent is at a pH of about 5.1 to about 7, about 5.5 to
about 7.5, about 6.5 to about 7.0 or about 6.8.
22. The method of any one of claims 14 to 21, wherein said
buffering agent is sodium cyclohexane diaminetetraacetate (CDTA),
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), acetic
acid or acetate (e.g. sodium acetate), citric acid or citrate,
malic acid, phthalic acid, succinic acid, histidine, pyrophosphoric
acid, maleic acid, cacodylic acid, .beta..beta.'-Dimethylglutaric
acid, carbonic acid or carbonate, 5(4)-Hydroxymethylimidazole,
glycerol 2-phosphoric acid, ethylenediamine, imidazole, arsenic
acid, phosphoric acid or phosphate, sodium acetate,
2:4:6-collidine, 5(4)-methylimidazole, N-ethylmorpholine,
triethanolamine, diethylbarbituric acid,
tris(hydroxymethyl)aminomethane (Tris),
3-(N-Morpholino)propanesulfonic acid; 4-morpholinepropanesulfonic
acid (MOPS), 2-morpholinoethanesulfonic acid (MES),
piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES),
N-[tris(hydroxymethypmethyl]-2-aminoethanesulfonic acid (TES),
4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), or combinations
thereof.
23. The method of any one of claims 14 to 22, wherein said
buffering agent is a phosphate buffer, a carbonate buffer, an
ethylenediamine buffer or an imidazole buffer.
24. The method of any one of claims 14 to 22, wherein the buffering
agent is CDTA or citric acid.
25. The method of any one of claims 14 to 24, wherein said sample
is a bodily fluid or a bodily tissue from a mammal.
26. The method of claims 25, wherein said mammal is a human or a
cow.
27. The method of claim 14, wherein said sample is from a human; a
non-human primate; livestock including cattle, pigs, sheep, goats
or domestic birds including chicken, turkey, pheasant, duck or
geese); game or wild animals including deer, elk, moose, fish,
birds or bears; laboratory or companion animals including non-human
primates, rodents including mice, rats, rabbits, guinea pigs,
gerbils or hamsters; dogs; cats; fish; snakes; lizards; turtles; a
horse; plants; plant parts; cell lines; soil microorganisms; sewage
microorganisms; or pathogenic microorganisms including virus,
bacteria or parasites.
28. The method of any one of claims 14 to 27, wherein the
composition stabilized said ribonucleic acid at room temperature
for at least about one day, two days, three days, four days, five
days, six days, one week, two weeks, three weeks, four weeks, five
weeks, six weeks, seven weeks or eight weeks, about one day to
about eight weeks, or greater than about eight weeks.
29. The method of any one of claims 14 to 27, wherein the
composition stabilizes said ribonucleic acid at room temperature
for about one day to about eight weeks at room temperature.
30. A RNA storage kit, comprising: a. a composition according to
any one of claims 1 to 13; and b. instructions for the use thereof.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Application Nos.
60/828,563; 60/866,985 and 60/949,778 the contents all of which are
hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The field of the invention generally relates to compositions
and methods for storage and/or isolation of ribonucleic acids from
bodily fluid(s), and/or secretion(s), (e.g., saliva, mucous),
and/or tissue(s).
BACKGROUND
[0003] The importance of detection and analysis of ribonucleic acid
(RNA) is becoming increasingly evident. For example, a large number
of pathogenic mammalian viruses (e.g. SARS-CoA, Influenza virus,
Measles virus, Rabies virus, Dengue fever virus, Respiratory
Syncytial Virus (RSV), HIV and Hepatitis A, C-E virus) have genomes
based on RNA rather than DNA. Detection and/or analysis of such RNA
are potentially of great importance, yet an accepted method that is
optimal for collecting, preserving/stabilizing, transporting and
extracting RNA has not yet been developed.
[0004] RNA is a labile compound and the widespread adoption for
routine use of RNA as an analyte in detection and analysis of RNA
has been limited because of its labile nature. The sugar-phosphate
backbone of RNA is particularly sensitive to breakdown
(degradation, hydrolysis) by alkaline solutions. It is also
sensitive to breakdown by acidic solutions. The pH of maximum
stability of RNA is generally assumed to be about neutral, but this
has not previously been determined precisely.
[0005] RNA can also be degraded enzymatically by endoribonucleases
(e.g., pancreatic ribonuclease). Ribonuclease activity has
previously been identified in human saliva (Bardon and Shugar,
1980), but the biochemical properties of this enzyme have not been
well characterized. Brandon and Shugar (1980) suggest that salivary
ribonuclease is pancreatic ribonuclease-like, but this has not been
established.
[0006] At least in part as result of its instability RNA is often
considered as an unsuitable analyte for diagnosis or detection. In
the case of RNA viruses, methods have been devised for detection
that do not require direct detection of RNA. For example, liquid
culturing systems are used to `grow up` sufficient quantities of
virus/bacteria to confirm a diagnosis. Bacterial infection is
typically diagnosed by direct staining and microscopic examination
of samples. Electron microscopy is also used to identify bacteria
and virus containing samples. In serology, diagnosis may be
accomplished by detection of antibodies directed against pathogens
(e.g. viruses, bacteria, parasites) in blood serum by employing
indirect fluorescent antibody testing and enzyme-linked
immunosorbent assays
[0007] Reverse transcriptase PCR (RT-PCR) procedures are sensitive
for detecting pathogens, and in some cases before the onset of
symptoms. Rapid viral diagnosis will become increasingly critical,
both for the control of epidemics and for the management of
patients with viral infections. Currently, an immunofluorescence
assay (IFA) is considered the "gold standard" for the detection of
SARS-CoA infection. However, this test requires culturing of
infectious SARS virus in laboratories with biosafety level 3
(BSL-3) facilities by well-trained technician personnel. Hence,
there is a need for a more convenient, economical, and low-risk
method for collecting and processing infectious clinical
specimens.
[0008] RNA can be extracted from most, if not all, cell types in
the human body (except erythrocytes) and from a variety of
cell-containing bodily fluids and/or secretions as well as tissues.
In some cases, it is also be desirable to be able to obtain RNA
from other sources, including feces, urine, cerebral spinal fluid,
animal tissues, bone marrow aspirates, plants, plant extracts,
microorganisms, virus, soil samples, sewage, wastewater, and/or
foodstuffs (including milk).
[0009] Typically, once a RNA-containing sample is collected, it
must either be frozen (e.g., with liquid nitrogen) or quickly
transported in the unfrozen state at 4.degree. C. to a laboratory
for extraction of RNA. The requirement for rapid transportation
and/or the requirement of freezing may be problematic in terms of
cost and storage space. Additionally, in the case of remote
locations and/or large-scale sample collection, rapid
transportation and/or freezing may not be feasible. Importantly,
rapid processing/testing of clinical samples may not be feasible
during an epidemic; back-logged samples will likely degrade over
time and/or under sub-optimal storage conditions. A simpler
procedure for collecting RNA in a form that would not require the
sample be frozen or transported immediately to a laboratory
including equipment such as freezers, refrigerators, centrifuges,
etc., would be desirable.
[0010] As noted above, there are a variety of cellular sources of
RNA. Cells from the oral cavity are conveniently obtained from
samples of saliva. Saliva can be collected `passively` by spitting
and/or `actively` with the aid of implements (e.g., swabs). Nasal
mucosal samples are conveniently obtained and are a rich source or
epithelial and immune cells (e.g., lymphocytes). This procedure is
not as invasive compared to, for example, taking of venous blood
and a simple procedure based on saliva would permit self-collection
by individuals with essentially no prior training. However, once
collected, the time that useable RNA can be recovered may be
limited because of the presence of ribonucleases in most tissues
and bodily fluids.
[0011] With the increasing use of nucleic acid-based testing in
human and veterinary medicine and in research, there is a need for
compositions and methods that would allow RNA to be reliably
recovered from bodily fluids and/or secretions and tissues.
Desirably, it should be possible to be able to store the collected
bodily fluid or bodily tissue at ambient temperature for prolonged
periods of time, for example several days or weeks. For example,
this would be advantageous where the bodily sample or bodily tissue
needs to be shipped to a distant location for purification and
analysis, especially in the absence of refrigeration or
freezing.
[0012] Cationic compounds, such as tetradecyltrimethylammonium
oxalate, have been used previously as a component in solutions used
in purification of nucleic acids. US20020146677 includes
tetradecyltrimethylammonium oxalate plus tartaric acid to stabilize
nucleic acid in blood. However, cationic compounds, including
tetradecyltrimethylammonium oxalate, have been found to be
unsatisfactory in terms of ease of use and long term stability of
RNA. It has been found that once the cationic detergent is bound to
nucleic acids, the nucleic acids are difficult to dissolve.
[0013] In addition, it would be desirable for the amount of RNA in
the collected sample to be sufficiently large to allow for the
detection of low copy number RNA species such as messenger RNA and
some viruses.
[0014] This background information is provided for the purpose of
making known information believed by the applicant to be of
possible relevance to the present invention. No admission is
necessarily intended, nor should be construed, that any of the
preceding information constitutes prior art against the present
invention.
SUMMARY OF THE INVENTION
[0015] An object of the present invention is to provide a
composition and method for prolonged storage of RNA from bodily
fluids and/or tissues at room temperature, which compositions and
methods further facilitate extraction of the RNA in as high a yield
and as nearly intact state as is possible.
[0016] In accordance with one aspect of the present invention there
is provided a composition for extracting and storing ribonucleic
acid from a sample such that the ribonucleic acid within said
sample remains stable at room temperature, said composition
comprises: an anionic detergent; and a buffering agent at a pH of
about 5 to about 8.2; wherein said composition stabilizes said
ribonucleic acid at room temperature.
[0017] In accordance with another aspect of the present invention
there is provided a method for preserving ribonucleic acid from a
biological sample comprising the steps of: a. obtaining the sample
from a subject; b, contacting said sample with a composition
comprising an anionic denaturing agent and a buffering agent at a
pH of about 5 to about 8.2 to form a mixture; c. storing the
mixture at room temperature; and d. heating the mixture at greater
than or about equal to 50.degree. C. prior to subsequent
processing, wherein said composition stabilizes said ribonucleic
acid at room temperature.
[0018] In accordance with another aspect of the present invention
there is provided a RNA storage kit, comprising: a. a composition
according to the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 is photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA from saliva stored in compositions of the present
invention;
[0020] FIG. 2 is a is photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA from saliva stored in compositions of the present invention
and stored in Oragene.TM.;
[0021] FIG. 3 is a photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA samples stored in compositions having a range of pH's;
[0022] FIG. 4 is a photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA combined with a cell-free fraction of saliva;
[0023] FIG. 5 is a photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA combined with a cell-free fraction of saliva over a range of
pH's;
[0024] FIG. 6 is photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA in saliva stored in a composition of the present invention
over a range of SDS concentrations;
[0025] FIG. 7 is a photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA in saliva when stored at room temperature compared to
37.degree. C., using a composition of the present invention;
[0026] FIG. 8 is a photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA in saliva stored at room temperature in a composition of the
present invention;
[0027] FIG. 9 is a photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA in saliva stored at room temperature in a composition of the
present invention;
[0028] FIG. 10 is a photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA samples stored in compositions of the present invention and
heated at various temperatures subsequent to storage at room
temperature;
[0029] FIG. 11 is a photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA samples stored in compositions of the present invention and
heated at various temperatures subsequent to storage at room
temperature;
[0030] FIG. 12 is a photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RNA samples stored at room temperature for 10-16 days at room
temperature in a composition of the present invention;
[0031] FIG. 13 is a photograph of an ethidium bromide-stained,
transilluminated agarose gel showing the results of electrophoresis
of RT-PCR products;
[0032] FIG. 14 is a photograph of ethidium bromide-stained,
transilluminated agarose gels showing the results of
electrophoresis of RNA samples from saliva stored at the indicated
temperature for 1 week (Panel A) and 8 weeks (Panel B); and
[0033] FIG. 15 is a diagram depicting steps that may be followed to
collect saliva from a subject.
DETAILED DESCRIPTION OF THE INVENTION
[0034] As will be described in more detail below, the present
invention relates to compositions and methods for prolonged
storage, and extraction, of ribonucleic acid (RNA) from bodily
fluids such as saliva, nasal secretions and/or tissues, wherein the
RNA in the resulting composition remains stable at room temperature
for extended periods of time.
[0035] The term "about", as used herein, refers to +/-10% of the
stated value or a chemical or obvious equivalent thereof.
[0036] The term "bodily fluid", as used herein, refers to a
naturally occurring fluid from a human or an animal, and includes,
but is not limited to saliva, sputum, serum, plasma, blood,
pharyngeal, nasal/nasal pharyngeal and sinus secretions, urine,
mucous, gastric juices, pancreatic juices, bone marrow aspirates,
cerebral spinal fluid, feces, semen, products of lactation or
menstruation, cervical secretions, vaginal fluid, tears, or
lymph.
[0037] The terms "bodily tissue" or "tissue", as used herein, refer
to an aggregate of cells usually of a particular kind together with
their intercellular substance that form one of the structural
materials of a plant or an animal and that in animals include
connective tissue, epithelium, mucosal membrane, muscle tissue, and
nerve tissue, and the like.
[0038] The term "Ct value", as used herein, is as defined in the
Operator Manual for our Rotor-Gene.TM. 6000 (real-time genetic
amplification detection system; manufactured by Corbett Life
Science) and refers to the fractional cycle number at the point
where the amplification curve crosses a threshold of detection. By
setting a threshold line and calculating the intersection with each
of the sample curves, the Ct values for each sample are
established. The threshold line is set in the exponential phase of
the run, significantly above the background level to avoid noise
and below the onset of signal plateau in later cycles.
[0039] The term "nucleic acid", as used herein, refers to a chain
of nucleotides, including deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), typically found in chromosomes, chromatin,
mitochondria, ribosomes, cytoplasm, nucleus, microorganisms (e.g.,
bacteria) or viruses.
[0040] The term "ribonucleic acid" or "RNA", as used herein, refers
to a wide range of RNA species, including, but not limited to high
molecular RNA, large and small ribosomal RNAs, messenger RNA,
pre-messenger RNA, small regulatory RNAs, RNA viruses (single and
double-stranded, positive stranded or negative stranded) and the
like. The RNA may be from a variety of sources, including, but not
limited to human, non-human, viral, bacterial, fungal, protozoan,
parasitic, single-celled, multi-cellular, in vitro, in vivo,
natural, and/or synthetic sources.
[0041] The term "primer", as used herein, refers to an
oligonucleotide acting as a starting point from which the synthesis
begins in the presence of a DNA template or a RNA template,
reagents for polymerization and so on. Although a primer is
preferably single-stranded, double-stranded primers may also be
used. When double-stranded primers are used, it is desirable to
convert them into their single-stranded forms before use in an
amplification reaction. A primer may be chemically or enzymatically
synthesized using well known methods, or may be isolated from an
organism.
[0042] The term "saliva", as used herein, refers to the secretion,
or combination of secretions, from any of the salivary glands,
including the parotid, submaxillary, and sublingual glands,
optionally mixed with the secretions from the numerous small
labial, buccal, and palatal glands that line the mouth.
[0043] The term "sputum", as used herein, refers to mucoid matter
contained in or discharged from the nasal or buccal cavity of a
mammal, including saliva and discharges from the respiratory
passages, including the lungs.
[0044] The term "subject", as used herein, refers to a variety of
organisms/sources, including, but not limited to human, non-human
mammals, other animal species, viral, bacterial, fungal, protozoan,
parasitic, single-celled, multi-cellular, in vitro, in vivo,
natural, and/or synthetic sources. Specific non-limiting examples
of suitable subjects include human and bovine sources. Specific
non-limiting examples include beef cattle, dairy cattle, sheep,
goats, hogs, poultry and horses. Specific non-limiting examples
also include companion animals, such as dogs, cats and the
like.
[0045] The term "prolonged storage" refers to storage for at least
about one day, two days, three days, four days, six days, one week,
two weeks, three weeks, four weeks, five weeks, six weeks, seven
weeks, or eight weeks, from about one day to about eight weeks, or
greater than about eight weeks.
Composition
[0046] The composition of the present invention is a composition
for extracting RNA from a bodily fluid or tissue and maintaining
the RNA contained therein stable at room temperature for prolonged
periods.
[0047] As will be discussed in more detail below, the composition
of the present invention includes an anionic detergent and a
buffer.
[0048] Selection of the specific components of the composition is
made based on various criteria, including, for example, efficacy
for stabilizing ribonucleic acid, cost, safety for the subject and
the laboratory worker, availability, and compatibility with
downstream applications. The choice of the components and their
concentration should be appropriate to stabilize the RNA in the
biological sample at room temperature.
[0049] The composition of the present invention permits storage of
the sample at room temperature and subsequent processing to isolate
and purify the RNA contained therein. The term "processing" as used
herein refers to mechanical or chemical steps used to isolate or
purify the ribonucleic acid from composition mixed with biological
sample.
[0050] A major cause of nucleic acid instability in biological
samples is the presence of deoxyribonucleases and ribonucleases.
Deoxyribonucleases and ribonucleases are enzymes that break down
DNA or RNA, respectively. Their main source in the digestive tract
is secretions of the pancreas, although these enzymes may also be
present in secretions and cells of the salivary gland and buccal
mucosa. In addition, microorganisms resident in the mouth or from
recently ingested foods may release deoxyribonucleases or
ribonucleases. Over time, the nucleic acid within a biological
sample (e.g., saliva) stored in water would be expected to degrade
or break down.
[0051] The compositions of the present invention provides
inhibition of nucleases, including ribonucleases, and chemical
stabilization of RNA. Ribonuclease inhibition and the ability to
store a ribonuclease-containing RNA sample at room temperature is
achieved through the use of an anionic detergent and a buffer
wherein the appropriate pH is maintained, followed by an incubation
step at about 50.degree. C. or above, prior to subsequent
processing of samples. Optionally, proteinase K is included in the
incubation step. An appropriate pH, as used herein, is (i) a pH at
which RNase activity is minimized or eliminated and (ii) a pH at
which RNA remains chemically stable.
Anionic Detergent
[0052] While not wishing to be bound by theory, it is thought that
action of deoxyribonucleases and ribonucleases is inhibited by
anionic detergents that destroy their complex structure,
particularly their catalytic sites. Hence, anionic detergents can
be included in the composition of the present invention.
Non-limiting examples of suitable anionic detergents include sodium
dodecyl sulfate (SDS), sodium sarcosinate (sarkosyl), lithium
dodecyl sulfate, sodium 1-octane sulfonic acid, and the like.
[0053] In accordance with a specific embodiment of the present
invention, the composition contains the anionic detergent SDS at a
concentration such as when it is mixed with saliva, the SDS is in
the range of about 0.5% to about 8%. In another example, the
composition contains the anionic detergent SDS at a concentration
such as when it is mixed with saliva, the denaturing agent is in
the range of about 1% to about 8% or 2% to about 8%.
[0054] In one example, the composition contains the denaturing
agent Sarkosyl at a concentration such that when it is mixed with
saliva, the concentration of sarkosyl is in the range of about 0.5%
to about 8%. In another example, the concentration of sarkosyl is
in the range of about 2% to about 4%.
Buffer
[0055] In accordance with one embodiment of the present invention,
the composition comprises an anionic detergent and a buffer to
maintain the pH within the range of 5-8.2. In accordance with
another embodiment of the present invention, the composition
comprises a denaturing agent and a buffer to maintain the pH within
the range of 5.1-7.0. In one example, the pH of the composition is
in the range of about 5.5 to about 7.5. In one example, the pH of
the composition is in the range of about 6.5 to about 7.0. In one
example, the pH of the composition is about 6.8. The pH of the
composition can be maintained at the desired pH using a buffer.
[0056] Non-limiting examples of suitable buffering agents include
sodium cyclohexane diaminetetraacetate (CDTA),
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), acetic
acid or acetate (e.g. sodium acetate), citric acid or citrate,
malic acid, phthalic acid, succinic acid, histidine, pyrophosphoric
acid, maleic acid, cacodylic acid, .beta..beta.'-Dimethylglutaric
acid, carbonic acid or carbonate, 5(4)-Hydroxymethylimidazole,
glycerol 2-phosphoric acid, ethylenediamine, imidazole, arsenic
acid, phosphoric acid or phosphate, sodium acetate,
2:4:6-collidine, 5(4)-methylimidazole, N-ethylmorpholine,
triethanolamine, diethylbarbituric acid,
tris(hydroxymethyl)aminomethane (Tris),
3-(N-Morpholino)propanesulfonic acid; 4-morpholinepropanesulfonic
acid (MOPS), 2-morpholinoethanesulfonic acid (MES),
piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES),
N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES),
4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), or combinations
thereof. Other examples include phosphate, carbonate,
ethylenediamine or imidazole buffers.
[0057] Additional non-limiting examples of suitable buffering agent
include buffering agents having a pKa at 25.degree. C. of from
about 4.7 to about 8.0.
[0058] In a specific example the buffer is CDTA. In another example
the buffer is citrate/citric acid.
[0059] The following are non-limiting examples of compositions
according to the present invention.
[0060] In accordance with a specific example, the composition
comprises 4% SDS, 50 mM CDTA adjusted to pH 6.2.
[0061] In accordance with a specific example, the composition
comprises 4% SDS, 50 mM CDTA adjusted to pH 6.6.
[0062] In accordance with a specific example, the composition
comprises: 16% SDS or 12% SDS or 8% SDS, 50 mM CDTA buffered to pH
6.2.
[0063] In accordance with a specific example, the composition
comprises 1% SDS, 50 mM CDTA adjusted to pH 6.6.
[0064] In accordance with a specific example, the composition
comprises 4% SDS, 50 mM citric acid buffered to pH 6.6.
[0065] In accordance with a specific example, the composition
comprises 4% Sarkosyl, 50 mM CDTA buffered to pH 6.6. In another
example the composition comprises 8% Sarkosyl, 50 mM CDTA buffered
to pH 6.6.
[0066] In accordance with a specific example, the composition
comprises 4% SDS, 50 mM LiCDTA, 250 mM LiCl adjusted to pH 6.8.
[0067] Surprisingly, it has been found that the composition of the
present invention can stabilize RNA, such as high molecular weight
RNA, present in biological samples such as sputum or saliva or
nasal, anterior nasal, and/or nasopharyngeal samples for prolonged
periods of time at room temperature. Notably, the RNA can be
purified using a variety of methods, and can be purified without a
requirement for phenol extraction, guanidinium salts, or any column
or binding matrix. The purified RNA is sufficiently pure to be used
directly in downstream applications such as, for example, the
preparation of complementary DNA (cDNA). However, phenol
extraction, guanidinium salts, or any column or binding matrix may
be used, if desired.
[0068] While not wishing to be bound by theory, in the case of the
specific example noted supra, it is believed that the anionic
detergent, for example SDS binds, denatures and inhibits salivary
ribonuclease(s) and the buffer, for example CDTA, keeps the pH of
the saliva sample neutral or slightly acidic. Buffering the sample
in a relatively narrow range helps to ensure the RNA is chemically
stable, despite the fact that ribonuclease(s), such as salivary
ribonuclease(s), are potentially active in this pH range. While
salivary RNA is stable for extended periods of time (e.g., weeks to
months) in this composition, it has been discovered that salivary
ribonuclease activity is robust in some samples may not be
permanently inactivated. Once the constraints of SDS have been
removed during subsequent processing/purification (e.g., if the
composition of the present application is diluted below a certain
concentration (e.g., below 0.5% SDS), the RNA within the sample may
be substantially degraded.
[0069] The Applicant has surprisingly discovered that heating the
sample at temperatures above about 50.degree. C. following storage
at room temperature and prior to subsequent processing allows the
RNA within the sample to be extracted in a substantially intact
form. Again, while not wishing to be bound by theory, it appears
that this heating step largely or completely inactivates salivary
ribonuclease activity while assisting in the liberation of RNA from
cells. The optional step of adding proteinase K to the biological
sample in this composition during the heating step, prior to
purification, helps digest proteins in the saliva and may also
contribute to the inactivation of salivary ribonuclease
activity.
[0070] In optimizing the components of one embodiment of the
composition of the present invention, the Applicant has determined
a pH range that, on the one hand, is optimal for minimizing
chemical degradation of the RNA and, on the other hand, permits SDS
to strongly inhibit the salivary ribonuclease activity. The
composition of the present application reduces ribonuclease
activity nearly completely while maintaining the chemical stability
of RNA.
[0071] In accordance with a specific example of the present
invention, the denaturing agent is not a guanidinium salt.
[0072] It has been found that RNA extracted and stored using the
compositions of the present invention is substantially intact,
suitable for RT-PCR analysis, and is recoverable from samples
without the aid of any nucleic acid-binding matrix such as
paramagnetic silica-coated beads or a silica-based membrane.
[0073] In one example, the method of the present invention promotes
recovery of intact, high molecular weight RNA.
Method
[0074] In accordance with another aspect of the present invention,
there is provided a method for storing RNA for prolonged periods at
room temperature.
[0075] Samples may be obtained from variety of sources including,
but not limited to humans, non-human primates, livestock (e.g.,
cattle, pigs, sheep, goats, domestic birds such as chicken, turkey,
pheasant, duck, geese), game and wild animals (e.g., deer, elk,
moose, fish, birds, bear), laboratory and companion animals (e.g.,
non-human primates, rodents such as mice, rats, rabbits, guinea
pigs, gerbils, hamsters), pigs, goats, sheep, dogs, cats, fish,
snakes, lizards, turtles, a horse and the like. Samples may also be
obtained from plants, cell lines, soil microorganisms, sewage
microorganisms, pathogenic microorganisms (e.g, virus, bacteria,
parasites) and the like.
[0076] In specific example, the sample is obtained from a human
source. In an alternate specific example, the sample is obtained
from a bovine source.
[0077] In a specific example, the RNA is within saliva. The method
comprises the steps of mixing a sample of saliva with the
composition of the present invention, then storing the
saliva-composition mixture at room temperature. In one example, The
method comprises the steps of mixing a sample of saliva with
approximately an equal volume of the composition of the present
invention, then storing the saliva-composition mixture at room
temperature. Prior to subsequent processing, the sample is heated
above about 50.degree. C. for a short period of time.
[0078] In an alternate specific example, the RNA is within a nasal,
anterior nasal and/or nasopharyngeal sample. The method comprises
the steps of mixing the nasal, anterior nasal and/or nasopharyngeal
sample with the composition of the present invention, then storing
the mixture at room temperature. The RNA is stable for at least
about one day and at least about four weeks. Prior to subsequent
processing, the sample is heated above 50.degree. C. for a short
period of time.
[0079] Advantages to the subject of providing a saliva sample or
nasal, anterior nasal and/or nasopharyngeal sample, rather than a
blood sample as a source of ribonucleic acid, include that subjects
typically prefer avoiding the discomfort, pain and apprehension
associated with phlebotomy. Additionally, and although use of a
pin-prick to obtain a drop of blood is sufficient to recover a
useable amount of DNA, the expected amount of RNA is too small to
be useable for most purposes. Saliva, sputum, nasal, anterior nasal
and/or nasopharyngeal samples have a further advantage of not
requiring specialized personnel for collection, thereby reducing
cost where mass sample collection is being carried out (e.g.,
during a epidemic/pandemic). However, it will be clear to the
skilled worker that while saliva is one source of RNA, other bodily
fluids, including blood, and bodily tissues, can be used. The
present invention is not intended to be limited to the collection
and storage of RNA obtained from sputum, saliva, nasal, anterior
nasal and/or nasopharyngeal samples.
[0080] To collect saliva from a subject it is preferred that the
mouth be rinsed before sampling. Food particles can introduce
foreign RNA and saliva transferred by kissing can be a source of
foreign human RNA or viral RNA. The mouth can be rinsed with about
50 ml of tepid water by vigorous swishing or by brushing with a
toothbrush without toothpaste. Unstimulated saliva is usually of
the mucinous type and is secreted at a slow rate. Stimulated saliva
(anticipation of tasty food, sweet or sour candy) is of the serous
(watery) type and secreted at a faster rate. After rinsing of the
mouth and waiting about 5 minutes for the mouth to clear of water,
the subject may spit a volume (for example, about 1-2 ml) of
saliva, preferably stimulated saliva, into the receiving tube.
Saliva flow can conveniently be stimulated with a few grains/pinch
of table sugar placed on top of the tongue, or any other such
saliva-stimulatory substance that does not interfere with RNA
stability or subsequent amplification.
[0081] Saliva may also be obtained from subjects such as infants,
young children and people with disabilities and/or illness that may
be unable to directly spit into a collection device. In this
instance, an implement (e.g., a swab etc.) is used to collect
saliva.
[0082] Saliva may also be obtained from non-human animals such as
livestock, companion animals and the like, which may be unable or
unwilling to directly spit into a collection device. In this
instance, an implement (e.g., a swab etc.) is used to collect
saliva.
[0083] To collect anterior nasal or nasopharyngeal samples from a
subject, a variety of implements may be used. Mucosal cells can be
scraped using rigid or flexible brushes, swabs, or plastic/wood
scrapers and cells may be flushed from the nasal cavity by
introducing a liquid (e.g., saline) and recovering the liquid. For
example, a rigid swab/brush can be placed in the anterior of the
nose and a flexible swab/brush into the posterior nasopharyngeal
cavity and used to collect mucosal secretions and to gently rub off
cells from the mucosal membrane. Samples collected with said liquid
and/or implement(s) can be delivered into a collection device
containing the composition of the present invention. In situations
where it is desirable to introduce a volume of said liquid that is
greater than the volume of the composition, a correspondingly
larger amount of composition of the present invention would be
provided. A cutting device (e.g. scissors) may be used to shorten
the length of a swab's/applicator's handle to permit closure of the
collection device. Alternately, swabs or brushes with handles that
snap under pressure, as well as swabs or brushes with a moulded
breakpoint in the handle/shaft, can be used to facilitate sample
collection. The `full length` handle facilitates the collection of
a sample and shortening of the swab/brush at the engineered break
point permits a better fit into the collection device. It is also
feasible to recover RNA from tissue samples taken from the nasal
cavity. Fresh tissue specimens/biopsies (e.g., normal nasal mucosa
or nasal polyp tissue) obtained from patients undergoing
rhinoplasty or endoscopic sinus surgery can be collected in the
composition of the present invention for subsequent RNA
isolation.
[0084] Methods of the invention are conveniently practiced by
providing the compositions used in such method in the form of a
kit. Such a kit preferably contains appropriate composition and may
include swabs to facilitate sample collection. At least one type of
positive control or standard may be provided that can be a nucleic
acid (DNA or RNA) template for demonstrating the suitability of the
sample for the detection of a target gene or nucleic acid sequence
(e.g. transcript). Such a kit preferably contains instructions for
the use there of.
[0085] Optionally, the kit includes a container, such as that
described in International PCT Application No. WO 03/104251, the
contents of which are incorporated herein by reference in its
entirety.
[0086] Optionally, the kit includes a container, such as that
described in U.S. Application Ser. No. 60/748,977, or
PCT/CA2006/002009 the contents both of which are incorporated
herein by reference in their entirety.
[0087] Optionally, the kit includes a collection assembly, such as
that described in PCT/US2007/64240, the contents of which are
incorporated herein by reference in its entirety.
[0088] Desirably, the container facilitates collection in the
field, without the requirement of a clinic or hospital, and is
sized to be mailed to a collection site and/or an analysis
site.
Applications
[0089] The compositions and methods of the present invention are
suitable for use in a wide range of applications.
[0090] With the continuing concern about respiratory
viral/bacterial epidemics/pandemics, the methods and compositions
of the present invention are expected to be valuable for the
wide-scale field collection, transport, storage, purification and
subsequent analysis in a diagnostic laboratory of biological
samples, such as nasal, nasopharyngeal, sputum and/or saliva
samples. Stability of said samples in the composition of the
present invention at room temperature is expected to be extremely
valuable in situations (e.g. epidemics) when laboratories are
overwhelmed with large numbers of patient samples.
[0091] Additionally, with the increased importance and requirement
of livestock monitoring and tracking, the compositions and methods
of the present application are suitable for collection, storage and
archiving of livestock samples for disease surveillance and
livestock monitoring. The compositions and methods of the present
application are also suitable for use with companion animals, such
as dogs, cats, and the like.
[0092] Saliva is expected to surpass blood as the sample of choice
for many clinical diagnostic and genetic tests. Generally, a
sputum, saliva, nasal, anterior nasal and/or nasopharyngeal sample
is a less hazardous specimen to collect and process than blood;
both saliva collection and nasal, anterior nasal and/or
nasopharyngeal collection is non-invasive or minimally-invasive for
the patient and can be easily collected on multiple occasions.
Generally, such sample collection does not require a skilled
technician.
[0093] Nasal, anterior nasal and nasopharyngeal samples have been
shown herein to be suitable for use with the methods and
compositions of the present invention.
[0094] The nasal cavity is considered part of the upper respiratory
tract. Nasal mucosa is a rich source of epithelial and immune cells
(e.g. lymphocytes). Airway mucosa (nasal cavity and lungs) is the
first site of exposure to inhaled pathogens (e.g. bacteria and
virus). T cells present in respiratory mucosa are believed to play
an important role in the regulation of mucosal immune responses to
foreign antigens (e.g. infectious microbial antigens) bombarding
the mucosal surface.
[0095] Respiratory viruses (e.g. Respiratory Syncytial Virus, RSV)
infect, replicate and are shed from respiratory mucosa; transmitted
to others via nose and mouth. RSV causes serious respiratory
infection in young children.
[0096] Nasal samples stabilized in the composition of the present
invention are useful for the analysis of gene expression profiles
of normal nasal mucosa and nasal polyp tissue to understand the
pathophysiology of a variety of rhinopathies/inflammatory diseases
(1).
[0097] Assessment of mucosal secretions/samples (saliva and nasal
samples) can provide important insights into mucosal (and systemic)
humoral and cellular responses induced by infection and/or
immunization (2, 9).
[0098] Both saliva and nasal samples collected into the composition
of the present invention can also be used for the
diagnosis/identification of pathogen(s), e.g. SARS-CoA, RSV,
measles virus, influenza virus, rabies virus, Dengue fever virus,
HIV, Hepatitis A, Hepatitis C-E virus, Mycobacterium, etc.
[0099] It is also increasingly important and desirable to detect
and/or analyze RNA from animals. The methods and compositions of
the present invention are suitable to use for the diagnosis and/or
identification of RNA viruses which infect livestock include.
Non-limiting examples include: foot-and-mouth disease virus (FMDV)
(which infects domesticated and wild ruminants and pigs; most
commonly spread of infection via inhalation of infectious droplets
originating in breath of infected animals); bovine leukosis virus
(BLV); bovine parainfluenza virus (e.g. Parainfluenza-3 virus,
PI-3); bovine respiratory syncytial virus (BRSV); porcine
reproductive and respiratory syndrome virus; vesicular stomatitis
virus; bovine viral diarrhea virus (aerosol infection); bovine
coronavirus (e.g. SARS-associated coronavirus (SARS-CoV)); BHV1
virus (bovine rhinotracheitis, respiratory disease); equine
arteritis virus (aerosol infection); Nipah virus (porcine
respiratory and neurologic syndrome); Porcine Respiratory Corona
Virus Infection (PRCV); rabies virus (mammals); Jaagsiekte Sheep
Retrovirus (contagious lung cancer in sheep); infectious bronchitis
virus (IBV) (poultry); avian pneumovirus (APV) (poultry); newcastle
disease virus (NDV) (poultry respiratory, nervous, and digestive
systems); Influenzavirus A (Avian influenza) subtype H5N1 ("avian
flu").
[0100] The methods and compositions of the present invention are
suitable for the stabilization and subsequent detection/analysis of
ribonucleic acids from bacteria, fungi, cells infected with virus,
isolated virus, tissue cultures, cell lines and bodily fluids
and/or tissues that are contaminated with, or suspected of being
contaminated with, virus, bacterial, protozoa, fungi and the like,
and combinations thereof.
[0101] Additionally, the compositions and methods of the present
invention are suitable for use in cytoplasmic, nuclear and/or
mitochondrial RNA stability and analysis, archival (e.g., banking)
of samples (fluids and/or tissues), tracking the source for the RNA
(e.g. placental/fetal versus maternal origin), tracing the lineage
of the ribonucleic acid (e.g. identification and characterization
of the infected source and subsequent transmission pathways of a
virus).
[0102] Additionally, desirably the compositions of the present
invention render pathogens (viruses and bacteria) non-infectious,
making for safe handling of clinical samples. This is particularly
the case once samples have been heated. In contrast, clinical
specimens collected in `traditional` sterile transport medium
(e.g., Viral transport media (VTM)--Annex 8. "Collecting,
preserving and shipping specimens for the diagnosis of avian
influenza A (H5N1) virus infection Guide for field operations,
October 2006. WHO) and suspected of containing a pathogen(s) must
be processed in biosafety level 3 or 4 containment facilities.
These facilities are not numerous and would be overwhelmed in the
event of an epidemic. Moreover, remote areas, e.g. some countries
in Africa, do not have biosafety level 3 or 4 containment
facilities/laboratories.
[0103] The compositions and methods of the present invention are
suited to collection and isolation of RNA from healthy and infected
individuals in the field, over wide geographical areas, which can
provide vital surveillance information for the prediction and
prevention of large-scale epidemics. Such samples would be
invaluable for identifying candidate vaccine strains and
identification of virus genotypes for molecular epidemiological
studies.
[0104] The compositions and methods of the present invention are
suitable for diagnosis of an infection in the early phase of an
illness (e.g., before seroconversion). This aspect is important for
1) managing patient care and improving disease outcome, and 2)
preventing/reducing transmission. The sensitivity of `traditional`
serological testing (as noted above) is too low for early detection
of infection. For instance, SARS-CoA can not be detected by this
traditional method until 14-28 days after the onset of symptoms
(e.g. fever). To address the need for early and rapid
identification of SARS-CoA, a reverse transcription (RT)-PCR-based
assay was advocated by the World Health Organization (WHO) and is
being routinely used for detecting virus-specific RNA (4, 5). More
recently, a loop-mediated isothermal amplification method for rapid
detection of severe acute respiratory syndrome coronavirus has been
developed (3). While significant progress has been made in
PCR-based diagnostic techniques, the success of these assays relies
heavily upon the collection of clinical specimens into sterile
transport medium and rapid transport to biosafety level 3/4
containment facilities at 4.degree. C. for immediate processing.
Typically, these samples are collected from patients already
admitted to hospital and precious time (days) is often `wasted`
replicating the virus in cell cultures, followed by monitoring for
cytopathic effects. As discussed above, the `traditional` methods
of sample collection into transport medium is inadequate during an
epidemic/pandemic when diagnostic laboratories would be overwhelmed
with samples. Laboratories have limited capacities for
refrigerating and/or freezing samples, so the degradation of
back-logged samples during an epidemic would be anticipated.
Samples would need to be recollected and precious time would be
wasted.
[0105] The compositions and methods of the present invention, in
combination with RT-PCR, are also suitable for use in the diagnosis
of subclinical disease. For instance, in the Netherlands, RT-PCR
tests have detected measles virus (MV) RNA in throat-swab specimens
from 5 days BEFORE until 12 days after the onset of rash (7). "Oral
fluid proved to be the most practical specimen for the simultaneous
detection of MV-specific IgM antibody and viral RNA. Viral RNA was
also detected in oropharyngeal specimens from 3 healthy contact
persons with serological proof of MV infection." Hence, samples
collected from the nasal and oral cavity can be used for the
diagnosis of clinical and subclinical MV infection. Similar
findings were reported by another group studying a measles outbreak
in the state of Victoria in Australia in 1999 (8). Of lymphocytes
(peripheral blood leukocytes), urine, throat swab, and serum
specimens, throat swab specimens were optimal/the preferred
specimen for detection of measles virus RNA during the first 2
weeks after the rash. In this case, the tip of the throat swab was
placed in 3 mL of sterile viral transport medium and was
transported to the laboratory at 4.degree. C.
[0106] While great strides have recently been made in the field of
diagnostic testing (i.e. RT-PCR tests), clinical specimens are
still collected into `sterile transport medium` (e.g., VTM
described supra) and thus extremely labile. Since RNA (human and/or
bacterial and/or viral transcript) in this type of medium will most
likely degrade in transit, the expression profile of the sample
once it reaches the diagnostic laboratory will not accurately
reflect the profile of the patient at the time of collection. There
is also a high probability that infectious agents within these
clinical samples are still infectious after transport to the
lab.
[0107] To gain a better understanding of the invention described
herein, the following examples are set forth. It should be
understood that these examples are for illustrative purposes only.
Therefore, they should not limit the scope of this invention in any
way.
EXAMPLES
Example 1
Protocol for Obtaining Saliva Samples from Human Subjects
[0108] The subject is instructed to wait for a period of 30-60
minutes before last eating. If possible, the subject will brush his
teeth (without using toothpaste). If possible, the subject will
rinse his/her mouth with 50 ml of water. The subject will be
requested to wait for 5-10 minutes to allow the mouth to clear of
water. For subjects able to spit, they will be instructed to spit
saliva into the special collection tube until the level of saliva
reaches the 1 or 2 ml mark. Waiting after last eating and rinsing
the mouth is desirable but not essential. Collection of saliva may
take several minutes. If the subject finds that he/she is unable to
deliver sufficient saliva, he/she will be given a few grains of
table sugar to chew or place on the top of their tongue, and told
not to be concerned if some of the sugar is spit into the tube. For
subjects unable to spit (e.g., infants, young children, individuals
with limitations/disabilities), an implement (e.g., swab, brush,
transfer pipette) may be used, along with sugar, for sample
collection. Similarly, a subject may be provided a liquid (e.g.,
mouthwash, water, saline) to gargle his/her mouth and throat or
saline to flush his/her nasal cavity. Samples collected with said
liquid would be delivered into the collection tube. In situations
where the saliva/sputum/nasal secretions have been substantially
diluted by said liquid, a correspondingly larger amount of
composition of the present invention would be provided.
[0109] Where two or more samples are to be taken from a subject for
purposes of comparing two compositions, the subject is asked to
deliver small amounts of saliva alternating between two or more
tubes until each tube is filled to the 1 ml mark. This is necessary
because the composition of saliva from the subject can vary during
the process of spitting.
[0110] When the required amount of saliva is collected, it is
immediately mixed with an equal volume of a composition. The
precise way in which this will be introduced will depend upon the
container design. Once the saliva is introduced and mixed with the
composition, the container is securely capped. The RNA-containing
sample can be maintained at room temperature for prolonged periods
of time. A portion of the RNA-containing sample in aqueous solution
can be used as a RNA template for a reverse transcription reaction
to produce complementary DNA (cDNA), which can then be used in a
PCR reaction.
Example 2
Comparison of the Present Composition to Oragene.TM.
Sample Collection
[0111] In this example, a single subject provided two sputum/saliva
samples (2 ml each) within a short period of time. One sample was
collected into a vial containing 2 ml of a composition of the
present invention comprising: 4% SDS, 50 mM CDTA, pH 6.6. Shortly
afterwards, the same subject provided a second sample into a vial
containing 2 ml of Oragene.TM. solution. Samples were shaken and
left at room temperature (RT) for 3 days before purification of
nucleic acids.
Methods
[0112] Nucleic acids were purified from this subject's saliva in
Oragene.TM. or in the composition of the present invention. A
portion of the subject's sample in Oragene.TM. was mixed with
proteinase K and heated at 50.degree. C. for 2 hours, without
(FIGS. 1 and 2) and with (FIG. 2) an additional, subsequent heating
at 90.degree. C. for 15 minutes. A portion of the subject's sample
in the composition was mixed with proteinase K, heated at
50.degree. C. for 2 hours and then at 90.degree. C. for 15 minutes
(FIG. 1). The inclusion of a short incubation at a temperature
above 50.degree. C. was found to be necessary for some saliva
samples to facilitate the extraction of intact, high molecular
weight RNA from saliva collected in the composition of the present
invention, but had no effect on saliva collected in
Oragene.TM..
[0113] Samples were then centrifuged briefly to remove insoluble
material and the DNA and RNA remaining in the supernatant was
precipitated with 2 volumes of 95% ethanol. The precipitate was
dissolved in an appropriate buffer containing 0.1% SDS and a 10
.mu.l aliquot (equivalent to about 10 .mu.l of undiluted saliva)
was analyzed by electrophoresis on a 0.9% agarose gel, and then
stained with ethidium bromide (1 .mu.g/mL) to visualize the DNA and
RNA (see FIGS. 1 and 2). A 10 .mu.L aliquot of each purified sample
was also treated with pancreatic ribonuclease before gel
electrophoresis to demonstrate that the high molecular weight
material stabilized in and extracted from the composition of the
present invention is, RNA (FIG. 1). Note that in FIG. 1, genomic
high molecular weight DNA present in the saliva sample is indicated
by an arrow (.fwdarw.), the characteristic ribosomal RNA doublet is
indicated by an asterisk (*), and low molecular weight RNA is
represented by a plus sign (+).
[0114] The sample order of FIG. 1 is as follows:
TABLE-US-00001 Lane Sample 1 1 Kb.sup.+ DNA ladder 2 RNA marker 3
Nucleic acids purified from saliva in Oragene .TM. 4 Blank lane 5
Purified sample (see lane 3) treated with ribonuclease prior to gel
electrophoresis. 6 Blank lane 7 Nucleic acids purified from saliva
in a composition of the present invention. 8 Blank lane 9 Purified
sample (see lane 7) treated with ribonuclease prior to gel
electrophoresis. 10 Blank lane 11 RNA marker 12 1 Kb.sup.+ DNA
ladder
[0115] The sample order of FIG. 2 is as follows.
TABLE-US-00002 90.degree. C., 15 min prior to Lane Sample nucleic
acid extraction 1 Lambda-HindIII DNA ladder 2 RNA marker 3 Nucleic
acids purified from saliva in no Oragene .TM. 4 Nucleic acids
purified from saliva in yes Oragene .TM.
Conclusions
[0116] This example demonstrates i) the ability of the composition
of the present invention to preserve salivary ribonucleic acid for
at least 3 days at room temperature and ii) the suitability of the
extraction/purification procedure for the recovery of substantially
intact ribonucleic acids. This example demonstrates the superiority
of the composition of the present invention over Oragene.TM., in
terms of RNA stability.
[0117] The preservation and extraction of intact, high molecular
weight RNA, indicative of ribosomal RNA (see double bands in lane
7, FIG. 1), suggests that other forms of RNA (such as messenger RNA
present in amounts too low to be detected by ethidium bromide
staining and transillumination), are also maintained in an intact
form in samples collected in the composition of the present
invention.
Example 3
Optimizing pH Range for Maintaining the Stability of Pure RNA
Derived from Sputum/Saliva
Methods
[0118] Pure RNA was diluted 6-fold into solutions buffered over a
wide range of pH values (pH 3.0, 5.0, 6.0, 7.0, 8.2 and 10.0):
Following a 16 hour period of incubation at 50.degree. C., to allow
partial hydrolysis of the phosphodiester backbone of the RNA to
occur, an aliquot of the RNA buffered to each pH value (noted
below) was analyzed by electrophoresis on a 0.9% agarose gel and
stained with ethidium bromide (FIG. 3).
[0119] The sample order of FIG. 3 is as follows:
TABLE-US-00003 Lane Sample 1 Lambda DNA HindIII digest 2 RNA marker
3 RNA, pH 3.0 4 RNA, pH 5.0 5 RNA, pH 6.0 6 RNA, pH 7.0 7 RNA, pH
8.2 8 RNA, pH 10.0
Conclusions
[0120] RNA is stable chemically at neutral to slightly acid pH, as
indicated by the preservation of the stained double bands
characteristic of ribosomal RNA seen in treatments at pH 5.0-8.2.
Note that a decrease in intensity of the upper band is expected
before a decrease in the lower band. While not wishing to be bound
by theory, this is likely due to larger species of ribosomal RNA
having a higher probability of suffering one phosphodiester
backbone cleavage compared to the shorter RNA species (lower band).
The characteristic banding pattern of the 2 ribosomal RNA species
disappears entirely at more extreme acid and basic pH values of 3.0
and 10.0, respectively.
Example 4
Demonstration of Potent RNASE Activity in the Cell-Free Fraction of
Saliva
Methods
[0121] Three subjects spit one millilitre of saliva into tubes
containing an equal volume of saline. Immediately thereafter, the
saliva samples and saline were mixed and subjected to a high speed
centrifugation to pellet the cells contained within the saliva. The
resultant supernatant or cell-free saliva fraction (CFSF) was
diluted and then mixed in increasing amounts with a fixed amount
(1.0 .mu.g) of pure RNA. The pure RNA mixed with CFSF was incubated
at 37.degree. C. for 30 min and then analyzed by agarose gel (1.0%)
electrophoresis. The gel was stained with ethidium bromide to
visualize the integrity of the RNA (FIG. 4).
[0122] The sample order of FIG. 4 is as follows:
TABLE-US-00004 Lane Sample 1 1 Kb.sup.+ DNA ladder 2 Pure RNA + no
CFSF 3 Pure RNA + 0.01 .mu.L CFSF 4 Pure RNA + 0.05 .mu.L CFSF 5
Pure RNA + 0.1 .mu.L CFSF 6 Pure RNA + 0.5 .mu.L CFSF 7 Pure RNA +
1.0 .mu.L CFSF
Conclusions
[0123] RNA is rapidly degraded when incubated with a traction of a
microlitre of cell-free saliva. Hence, potent ribonuclease activity
exists in the extracellular fraction of saliva.
[0124] This example also demonstrates the existence of considerable
variability in salivary ribonuclease among between subjects.
Compared to subjects 1 and 2, a much smaller volume of CFSF from
subject 3 was required to degrade an equal amount of RNA.
Example 5
Optimal pH for Salivary Ribonuclease Activity
Methods
[0125] To determine the pH range where salivary ribonuclease
exhibits activity, an equivalent of 0.1 .mu.L, CFSF (Subject 2,
example 4) was mixed with 1 mg of pure RNA buffered to pH 5.1, 5.5,
6.0, 6.5, 7.0, 7.5, 8.1 and 8.6. Pure RNA, in the absence of CFSF
(lane 10), was included in this example to illustrate the state or
intact nature of the RNA utilized in this example. Following
incubation at 37.degree. C. for 30 min, the samples were analyzed
by agarose gel (0.9%) electrophoresis and stained with ethidium
bromide (FIG. 5). Indications of RNA degradation include 1) the
disappearance of one or both ribosomal RNA subunits, the distinct
double bands at the mid-point of the gel, 2) the appearance of an
elongated smear of ethidium bromide-stained material, and/or 3) the
hastened mobility of ethidium bromide-stained material on the gel
with respect to the control RNA marker.
[0126] The sample order of FIG. 5 is as follows:
TABLE-US-00005 Lane Sample 1 1 Kb.sup.+ DNA ladder 2 Pure RNA +
CFSF, pH 5.1 3 Pure RNA + CFSF, pH 5.5 4 Pure RNA + CFSF, pH 6.0 5
Pure RNA + CFSF, pH 6.5 6 Pure RNA + CFSF, pH 7.0 7 Pure RNA +
CFSF, pH 7.5 8 Pure RNA + CFSF, pH 8.1 9 Pure RNA + CFSF, pH 8.6 10
Pure RNA + no CFSF
Conclusions
[0127] Pure RNA, buffered at pH>6.5, was rapidly degraded (lanes
6-9) upon the addition of 0.1 .mu.L of cell-free saliva. Between pH
5.1 and 6.5, pure RNA remained intact in the presence of cell-free
saliva. These findings suggest that ribonuclease endogenous to
saliva displays optimal enzymatic activity at neutral (pH 7.0) to
slightly alkaline pH. There was no significant ribonuclease
activity detected in acid conditions (lanes 2-5).
Example 6
Stability of RNA Over a Range of SDS Concentration
Methods
[0128] Examples of RNA extracted from saliva taken from 5 subjects
into one of 3 compositions of the present invention are shown (FIG.
6). Saliva was collected and immediately mixed with an equal volume
of the indicated composition. The compositions contained SDS
(sodium dodecyl sulphate, at the concentration indicated in the
table below) and were buffered at pH 6.2 with 50 mM CDTA (sodium
salt of cyclohexane diaminetetraacetic acid). Each sample of saliva
was mixed with an equal volume of the indicated composition and
stored for 3 weeks at room temperature. To examine the RNA
contained in each sample, a 50 .mu.l aliquot was removed and heated
at 90.degree. C. for 15 min, then diluted 5-fold. Proteinase K was
added to each diluted aliquot and then incubated at 50.degree. C.
for 1 hour to allow the protease to digest proteins. After cooling
to room temperature, 10 .mu.l of a 2.5 M solution of KCl was added
to precipitate the SDS; the sample was then centrifuged to remove
the precipitated SDS. Cold 95% ethanol (2 volumes) was added to the
clear supernatant to precipitate the nucleic acids. After standing
for 1 hour at -20.degree. C., the precipitated nucleic acids were
dissolved in 25 .mu.l of a dilute buffer. 10 .mu.l of this solution
(equivalent to about 10 .mu.l of the original saliva) was applied
to a 0.9% agarose gel and subjected to electrophoresis for 1 hour.
The gel was stained with ethidium bromide and photographed under
transillumination. In each sample lane, the upper band is genomic
DNA. The 2 bands in the middle of the gel represent ribosomal RNA
that was present in saliva and preserved by the indicated
compositions.
[0129] The sample order of FIG. 6 is as follows:
TABLE-US-00006 Amount of SDS in composition Lane Sample (%) 1 1
Kb.sup.+ DNA ladder 2 RNA marker 3 Subject 1 16 4 Subject 1 12 5
Subject 1 8 6 Subject 2 16 7 Subject 2 12 8 Subject 2 8 9 Subject 3
16 10 Subject 3 12 11 Subject 3 8 12 Subject 4 16 13 Subject 4 12
14 Subject 4 8 15 Subject 5 16 16 Subject 5 12 17 Subject 5 8 18
RNA marker
Conclusions
[0130] These data demonstrate that RNA in saliva is stable for at
least 3 weeks at room temperature when mixed 1:1 with compositions
including a range of anionic detergent concentrations (8-16% SDS),
buffered to a slightly acidic pH (6.2). These data also illustrate
considerable variability between subjects in the amount of RNA
present in saliva.
Example 7
Stability of RNA in Saliva Using the Composition of the Present
Invention at Room Temperature and 37.degree. C.
[0131] In this example, the composition comprised 16% SDS, 50 mM
CDTA, buffered at pH 6.2. Saliva samples were mixed 1:1 with the
composition and stored for 7 days at either room temperature (RT)
or 37.degree. C. Incubating samples at 37.degree. C., compared to
RT, is expected to accelerate the degradation of RNA by salivary
ribonuclease, if present, should it retain activity in the
composition of the present invention. The samples were heated at
90.degree. C. for 15 min, then diluted 5-fold and incubated at
50.degree. C. for 1 hour with proteinase K. SDS was precipitated
with potassium chloride and, after the precipitate was removed by
centrifugation, nucleic acids were precipitated from the
supernatant with 2 volumes of cold 95% ethanol. A portion of each
precipitated nucleic acid sample was analyzed by agarose gel
electrophoresis, stained with ethidium bromide and photographed
under transillumination (FIG. 7).
[0132] The sample order of FIG. 7 is as follows:
TABLE-US-00007 Lane Sample 1 RNA marker 2 Subject 1-RT 3 Subject
2-RT 4 Subject 3-RT 5 Subject 4-RT 6 Subject 1-37.degree. C. 7
Subject 2-37.degree. C. 8 Subject 3-37.degree. C. 9 Subject
4-37.degree. C. 10 RNA marker
Conclusions
[0133] This example demonstrates the efficacy of the composition of
the present invention for stabilizing RNA in samples of saliva.
After 1 week at 37.degree. C., saliva samples from 4 subjects
showed no appreciable degradation of high molecular weight RNA.
Example 8
Testing a RNA-Stabilizing Composition Containing a Different
Buffer
[0134] In this example, sodium citrate/citric acid buffer (pK.sub.a
6.4, 50 mM) was substituted for CDTA, which was used to buffer the
pH in previous compositions. As in other examples, saliva was
collected from two subjects and mixed 1:1 with the composition (4%
SDS, 50 mM citric acid buffered to pH 6.6). Samples were then
stored at room temperature for 3 weeks. To extract RNA present in
the saliva/composition mixture, the samples were incubated at
50.degree. C. for 1 hour with proteinase K, heated at 90.degree. C.
for 15 min, SDS was precipitated with potassium chloride and, after
the precipitate was removed by centrifugation, nucleic acids were
precipitated from the supernatant with 2 volumes of cold ethanol.
To confirm the extracted material was RNA, a portion of the
precipitated nucleic acids was treated with pancreatic ribonuclease
(RNase). RNase-treated and -untreated extracts were then resolved
by agarose gel electrophoresis and stained with ethidium bromide as
before (FIG. 8). Note that part of the rapidly migrating,
RNase-resistant material in lanes 5 and 9 is likely DNA, which has
been denatured and partially degraded by the period of heating at
90.degree. C.
[0135] The sample order of FIG. 8 is as follows:
TABLE-US-00008 Lane Sample 1 Lambda DNA HindIII digest 2 RNA marker
3 Subject 1 4 Blank lane 5 Subject 1 + RNase 6 Blank lane 7 Subject
2 8 Blank lane 9 Subject 2 + RNase 10 Lambda DNA HindIII digest
Conclusions
[0136] This example demonstrates the suitability of substituting
citric acid for CDTA to buffer the pH of the composition.
Example 9
Testing the RNA-Stabilizing Solution Using a Different Anionic
Detergent
[0137] In this example, Sarkosyl (N-Lauroylsarcosine sodium salt or
sodium lauroyl sarcosinate) was substituted for SDS. Saliva was
collected from 3 subjects and mixed 1:1 with two compositions, i)
4% Sarkosyl, 50 mM CDTA buffered to pH 6.6 and ii) 8% Sarkosyl, 50
mM CDTA buffered to pH 6.6. Samples were then stored at room
temperature for 3 weeks. To extract RNA present in the
saliva/composition mixture, a portion of the samples were incubated
at 50.degree. C. for 1 hour with proteinase K, heated at 90.degree.
C. for 15 min, treated with potassium chloride and centrifuged.
Nucleic acids remaining in the supernatant were precipitated with 2
volumes of cold ethanol. To confirm the extracted material was RNA,
a portion of the precipitated nucleic acids was treated with
pancreatic ribonuclease (RNase). RNase-treated and -untreated
extracts were then resolved by agarose gel electrophoresis and
stained with ethidium bromide (FIG. 9).
[0138] The sample order of FIG. 9 is as follows:
TABLE-US-00009 Lane Sample 1 Lambda DNA HindIII digest 2 RNA marker
3 Subject 1, 4% Sark 4 Subject 1, 8% Sark 5 Subject 2, 4% Sark 6
Subject 2, 8% Sark 7 Subject 3, 4% Sark 8 Subject 3, 8% Sark 9
Blank lane 10 Subject 1, 4% Sark + RNase 11 Subject 1, 8% Sark +
RNase 12 Subject 2, 4% Sark + RNase 13 Subject 2, 8% Sark + RNase
14 Subject 3, 4% Sark + RNase 15 Subject 3, 8% Sark + RNase 16
Lambda DNA HindIII digest
Conclusions
[0139] This experiment shows that Sarkosyl, another strong anionic
detergent, can be substituted for SDS in the composition of the
present invention. Examples 6 and 0.9 demonstrate that anionic
detergents, with appropriate buffering agents, effectively
stabilize RNA in saliva.
Example 10
Demonstration that a Step Involving Brief Heating Above 50.degree.
C. is Beneficial in the Extraction of RNA from Saliva
[0140] In this example, the composition comprised 4% SDS, 50 mM
CDTA, buffered at pH 6.6. Saliva sample from one subject was mixed
1:1 with the composition and incubated for 6 days at room
temperature. To extract RNA present in the saliva/composition
mixture, the samples were first incubated at 50.degree. C. for 1
hour with proteinase K to digest protein. Aliquots were taken and
heated for 15 minutes at 50.degree. C., 55.degree. C., 60.degree.
C., 65.degree. C., 70.degree. C., 75.degree. C., 80.degree. C.,
85.degree. C. or 90.degree. C. SDS was precipitated from heated
aliquots with potassium chloride and, after the precipitate was
removed by centrifugation, nucleic acids were precipitated from the
supernatant with 2 volumes of cold ethanol. A portion of each
precipitated nucleic acid sample was analyzed by agarose gel
electrophoresis, stained with ethidium bromide and photographed
under transillumination (FIG. 10).
[0141] The sample order of FIG. 10 is as follows:
TABLE-US-00010 Lane Sample 1 1 Kb.sup.+ DNA ladder 2 RNA marker 3
50.degree. C. 4 55.degree. C. 5 60.degree. C. 6 65.degree. C. 7
70.degree. C. 8 75.degree. C. 9 80.degree. C. 10 85.degree. C. 11
90.degree. C. 12 RNA marker
Conclusions
[0142] In this example, 15 minutes of heating at temperatures above
50.degree. C. and as high as 90.degree. C. improved the yield of
high molecular weight RNA extracted from this saliva/composition
sample, compared to 50.degree. C. alone. These results also show
that the extracted RNA is not significantly degraded by a 15 minute
heating step at temperatures up to 90.degree. C.
Example 11
Demonstration that a Step Involving Brief Heating at 90.degree. C.
is Beneficial in the Extraction of RNA from Saliva
[0143] In this example, the composition comprised 4% SDS, 50 mM
CDTA, buffered at pH 6.6. Saliva samples from 2 subjects were mixed
1:1 with the composition and incubated for 4 days at room
temperature. To extract RNA present in the saliva/composition
mixture, the samples were first incubated at 50.degree. C. for 1
hour with proteinase K to digest protein. Aliquots were taken and
heated at 90.degree. C. for 0, 5, 15, 30 or 60 min, then diluted
4-fold with water and incubated for an additional 18 hours at room
temperature. Dilution of the sample permitted testing the
efficiency of the heating step by decreasing the concentration of
SDS to a level that would normally permit RNase activity. SDS was
precipitated with potassium chloride and, after the precipitate was
removed by centrifugation, nucleic acids were precipitated from the
supernatant with 2 volumes of cold ethanol. A portion of each
precipitated nucleic acid sample was analyzed by agarose gel
electrophoresis, stained with ethidium bromide and photographed
under transillumination FIG. 11; subject 1, left panel; subject 2,
right panel).
[0144] The sample order of FIG. 11 is as follows:
TABLE-US-00011 Lane Time (min) at 90.degree. C. 1 0 2 5 3 15 4 30 5
60 6 RNA marker
Conclusions
[0145] In this example, 5-15 minutes of heating at 90.degree. C.
improved the yield of intact RNA extracted from 2 subjects'
samples, compared to unheated samples. Evidence of RNA degradation
was observed in samples heated at 90.degree. C. for 30 and 60
minutes.
[0146] Additionally, the recovery of substantially intact ribosomal
RNA following heating, dilution and then further incubation of
samples, suggests that the composition of the present invention and
the extraction protocol result in purified material with diminished
ribonuclease activity.
Example 12
Human mRNA is Extracted and Purified from Samples Collected in the
Present Composition
[0147] In this example, it is demonstrated that Reverse
Transcriptase-Polymerase Chain Reaction (RT-PCR) can be used to
detect human-specific messenger RNA in RNA recovered from the
saliva of six subjects collected in the composition of the present
invention.
Methods
[0148] Saliva samples from 6 subjects were mixed 1:1 with the
composition (4% SDS, 50 mM CDTA, buffered at pH 6.6) and stored for
10-16 days at room temperature. Following storage at room
temperature, a portion of each saliva/composition mixture was
heated at 50.degree. C. for 1 hour with proteinase K, followed by
90.degree. C. for 15 min. SDS was precipitated with potassium
chloride and, after the precipitate was removed by centrifugation,
nucleic acids were precipitated from the supernatant with 2 volumes
of cold 95% ethanol. A portion of each precipitated nucleic acid
sample was analyzed by agarose gel electrophoresis, stained with
ethidium bromide and photographed under transillumination (FIG.
12).
[0149] A modified version of the Schmidt-Tannhauser procedure was
used to estimate the total amount of RNA in the saliva samples
collected from the same 6 subjects in the composition of the
present invention (FIG. 12). In brief, a portion of each sample was
treated with sodium hydroxide to selectively degrade RNA to oligo-
or mono-nucleotides (rendering it non-precipitable by cold
hydrochloric acid), leaving undegraded DNA precipitable with cold
HCl. In this way, DNA and RNA are separated and can be quantified
by absorbance measurements at 260 nm.
[0150] To demonstrate that human messenger RNA (mRNA) is stabilized
and recovered from these saliva samples, a portion of purified
sample from each subject served as template in RT-PCR analysis
using human-specific primers for .beta.2-microglobulin messenger
RNA (FIG. 13). Specifically, a portion of precipitated nucleic acid
sample from each subject (FIG. 12) was treated with DNase to digest
DNA. The DNA-free sample was then mixed with random hexanucleotide
primers and M-MVL reverse transcriptase to synthesize complementary
DNA (cDNA). Finally, the cDNA was diluted and mixed with
human-specific primers (.beta.2-microglobulin-forward 5'
cgctactctctctttctggc and .beta.2-microglobulin-reverse 5'
aacttcaatgtcggatggat) and Taq DNA polymerase for conventional PCR
(Table I); SybrGreen was added for quantitative real-time PCR
analysis (Table II). The Ct value, as shown in FIG. 15, is
inversely proportional to the amount of messenger RNA in the
sample. RNA purified from a human colon carcinoma cell line,
HCT-116, served as a positive control for RT-PCR analysis. Negative
controls include reactions in which no M-MVL RT or cDNA was
added.
TABLE-US-00012 TABLE I Subject Estimated amount (.mu.g) of RNA in
total sample 1 99.8 2 43.6 3 337.5 4 330.5 5 96.8 6 96.8
[0151] The sample order of FIG. 13 is as follows:
TABLE-US-00013 Source of RNA for RT-PCR with primers specific for
human .beta.2- Lane microglobulin (140 bp) L 1 Kb.sup.+ DNA ladder
1 Subject 1 2 Subject 2 3 Subject 3 4 Subject 4 5 Subject 5 6
Subject 6 7 No RT Negative control 8 No template (cDNA): Negative
control 9 HCT116 cell line: Positive control
TABLE-US-00014 TABLE II Real time-PCR with primers specific for
human .beta.2-microglobulin. Sample Ct value Subject 1 26.06
Subject 2 20.72 Subject 3 23.62 Subject 4 23.9 Subject 5 24.55
Subject 6 29.56 No RT >40 HCT116 cell line: 20.24 Positive
control No template (cDNA): >40 Negative control
Conclusions
[0152] This example demonstrates that human messenger RNA in saliva
is stabilized by the composition of the present invention and the
extracted material is suitable for RT-PCR analysis.
Example 13
Preservation, Release and Purification of Viral RNA Mixed with
Saliva Collected in the Composition of the Present Invention
[0153] In this example, it is demonstrated that Reverse
Transcriptase-Polymerase Chain Reaction (RT-PCR) can be used to
detect viral RNA in total RNA recovered from saliva collected in
the composition of the present invention.
Methods
[0154] Two saliva (1 mL) samples from subject 1 were `spiked` with
4.25.times.10.sup.9 pfu (plaque-forming units) of Vesicular
Stomatitis Virus (VSV) and incubated at room temperature for 5 min
prior to being mixed 1:1 with 4% SDS, 50 mM CDTA, buffered at pH
6.6. A 0.5 mL aliquot from each saliva/composition sample was
removed and heated at 50.degree. C. for 1 hr with proteinase K,
followed by 90.degree. C. for 15 mM. SDS was precipitated with
potassium chloride and, after the precipitate was removed by
centrifugation, nucleic acids were precipitated from the
supernatant with 2 volumes of cold 95% ethanol. Each precipitate
was re-dissolved in an appropriate buffer (100 .mu.l of CBS
containing 0.1 M NaCl) and then nucleic acids were re-precipitated
by adding 200 .mu.l of cold ethanol and incubating for 40 min at
-20.degree. C. Precipitated nucleic acids were re-dissolved in 50
.mu.l of water with ribonuclease inhibitor. One sample from subject
1 was kept at room temperature for 4 weeks prior to purifying the
nucleic acids as described above. A 2 mL saliva sample from subject
2 was mixed 1:1 with the composition of the present invention, then
spiked with 5.0.times.10.sup.7 pfu of VSV. After incubation at room
temperature for 18 hours, RNA was extracted from a 0.5 mL aliquot
by incubating at 80.degree. C. for 40 min; subsequent steps in
purification were as described above.
[0155] To demonstrate that viral RNA is stabilized and can be
recovered from these saliva samples, a portion of the purified
nucleic acids was used as template in RT-PCR, primed with random
hexanucleotide. Specifically, a portion of the sample was mixed
with random hexanucleotide primers and M-MVL reverse transcriptase
to synthesize complementary DNA (cDNA). A portion of the cDNA was
mixed with virus-specific primers (VSV-forward 5' ggattattccctctgcc
and VSV-reverse 5' gttccctttctgtggtag), Taq DNA polymerase and
SybrGreen and analysed by quantitative real-time PCR analysis. The
Ct value, as shown in FIGS. 16-18, is inversely proportional to the
amount of virus template RNA in the sample. Plasmid DNA encoding
the virus sequences (pVSV) served as a positive control for RT-PCR
analysis. Negative controls include reactions in which no M-MVL RT
(-RT) or no cDNA was added. RT-PCR results are shown for three
separate experiments (Tables III-V).
TABLE-US-00015 TABLE III Viral particles added (assuming 100%
efficiency in Ct stability, extraction and cDNA Sample/Source of
viral RNA value synthesis) pVSV 11.3 15 ng plasmid DNA pVSV 17.2 3
ng plasmid DNA NTC (no template control) 39.9 VSV in composition
with poly(A) 19.4 2.81 .times. 10.sup.9 pfu RNA carrier VSV in
saliva/composition (subject 29.8 1.48 .times. 10.sup.8 pfu 1,
sample 1) after 1 day at room temperature VSV in saliva/composition
(subject 29.5 1.48 .times. 10.sup.8 pfu 1, sample 2) VSV in
saliva/composition (subject 25.4 1.06 .times. 10.sup.9 pfu 1,
sample 1) after 4 weeks at room temperature VSV in
saliva/composition (subject 29.8 1.25 .times. 10.sup.6 pfu 2) after
1 day at room temperature -RT using VSV in saliva/ >40.0 1.25
.times. 10.sup.6 pfu composition (subject 2) after 1 day at room
temperature
TABLE-US-00016 TABLE IV Viral particles added (assuming 100%
efficiency in Ct stability, extraction and cDNA Sample/Source of
viral RNA value synthesis) pVSV 13.0 15 ng plasmid DNA NTC >45.0
VSV in composition with polyA 15.4 2.81 .times. 10.sup.9 pfu RNA
carrier VSV in saliva/composition (subject 27.5 1.06 .times.
10.sup.9 pfu 1) after 4 weeks at room temperature VSV in
saliva/composition (subject 30.5 1.25 .times. 10.sup.6 pfu 2) after
1 day at room temperature -RT using VSV in saliva/ 40.4 1.25
.times. 10.sup.6 pfu composition (subject 2) after 1 day at room
temperature
TABLE-US-00017 TABLE V Viral particles added (assuming 100%
efficiency in Ct stability, extraction and cDNA Sample/Source of
viral RNA value synthesis) pVSV 16.6 15 ng plasmid DNA NTC 39.3 VSV
in composition containing 19.3 2.81 .times. 10.sup.9 pfu polyA RNA
carrier VSV in saliva/composition (subject 26.4 1.48 .times.
10.sup.8 pfu 1, sample 1) after 1 day at room temperature VSV in
saliva/composition (subject 32.1 1.06 .times. 10.sup.9 pfu 1,
sample 1) after 4 weeks at room temperature VSV in composition
alone with 33.0 4.25 .times. 10.sup.9 pfu LPA (linear
polyacrylimide) VSV in Oragene alone with LPA 39.4 4.25 .times.
10.sup.9 pfu -RT using VSV in composition 36.6 4.25 .times.
10.sup.9 pfu containing polyA RNA carrier
Conclusions
[0156] This example demonstrates that viral RNA in saliva is
stabilized by the composition of the present invention and the
extracted material is suitable for RT-PCR analysis.
Example 14
Stability of RNA in Saliva at Room Temperature for 8 Weeks
[0157] In this example, the composition comprised 4% SDS, 50 mM
LiCDTA, 250 mM LiCl, adjusted to pH 6.8. Saliva samples (2 mL) from
10 subjects were collected and mixed 1:1 with the composition.
Immediately after collection, the samples were vigorously shaken
and 100 .mu.L aliquots were removed. Individual aliquots were
stored at room temperature (RT), 4.degree. C., and -20.degree. C.
Following 1 week and 8 weeks storage at RT, 4.degree. C., and
-20.degree. C., the samples were analysed. To extract RNA from the
saliva/composition mixture, the aliquots were heated at 50.degree.
C. for 1 hour in the presence of proteinase K, then at 90.degree.
C. for 15 min. SDS was precipitated with potassium chloride and,
after the precipitate was removed by centrifugation, nucleic acids
were precipitated from the supernatant with 2 volumes of cold 95%
ethanol. A portion of each precipitated nucleic acid sample was
analyzed by agarose gel electrophoresis, stained with ethidium
bromide and photographed under transillumination (FIGS. 14A and
14B).
[0158] To demonstrate that human messenger RNA (mRNA) is stabilized
and can be recovered from these saliva samples after 1 and 8 weeks
at room temperature, a portion of purified sample from each subject
was used as template in Reverse Transcriptase-PCR (RT-PCR) analysis
using primers specific for human 18S ribosomal RNA. Specifically, a
portion of precipitated nucleic acid sample from each subject was
treated with DNase to remove DNA prior to the Reverse Transcriptase
step. The DNA-free sample was then mixed with random hexanucleotide
primers and M-MVL reverse transcriptase to synthesize complementary
DNA (cDNA). Finally, the cDNA was diluted and mixed with
human-specific primers (human 18S-165 forward 5'
gtggagcgatttgtctggtt and human 18S-165 reverse 5'
ggacatctaagggcatcacag), Taq DNA polymerase and SybrGreen for
quantitative real-time PCR analysis. The Ct (Crossing Threshold)
values, as shown in FIG. 20, are inversely proportional to the
amount of 18S RNA in the sample. RNA purified from a human colon
carcinoma cell line, HCT-116, served as a positive control for
RT-PCR analysis. Negative controls include reactions in which no
M-MVL RT (-RT) or no cDNA/template was added.
[0159] The sample order of FIG. 14 is as follows:
TABLE-US-00018 Lane Sample Storage condition 1 Subject 1 RT 2
Subject 1 4.degree. C. 3 Subject 1 -20.degree. C. 4 Subject 2 RT 5
Subject 2 4.degree. C. 6 Subject 2 -20.degree. C. 7 Subject 3 RT 8
Subject 3 4.degree. C. 9 Subject 3 -20.degree. C. 10 Subject 4 RT
11 Subject 4 4.degree. C. 12 Subject 4 -20.degree. C. 13 Subject 5
RT 14 Subject 5 4.degree. C. 15 Subject 5 -20.degree. C. 16 Subject
6 RT 17 Subject 6 4.degree. C. 18 Subject 6 -20.degree. C. 19
Subject 7 RT 20 Subject 7 4.degree. C. 21 Subject 7 -20.degree. C.
22 Subject 8 RT 23 Subject 8 4.degree. C. 24 Subject 8 -20.degree.
C. 25 Subject 9 RT 26 Subject 9 4.degree. C. 27 Subject 9
-20.degree. C. 28 Subject 10 RT 29 Subject 10 4.degree. C. 30
Subject 10 -20.degree. C.
TABLE-US-00019 TABLE VI Real time-PCR with primers specific for
human 18S ribosomal RNA. 1 week 8 week 8 week aliquot 1 week
aliquot aliquot aliquot -RT Saliva Sample Ct value -RT Ct value Ct
value Ct value Subject 1 13.7 27.9 16.5 28.6 Subject 2 12.6 26.7
13.0 29.1 Subject 3 13.9 26.5 13.8 27.8 Subject 4 13.7 28.4 13.3
28.5 Subject 5 14.9 28.6 13.6 29.1 Subject 6 15.5 26.7 13.6 22.1
Subject 7 19.2 29.2 18.9 27.2 Subject 8 17.6 30.7 18.3 23.9 Subject
9 13.0 22.0 17.1 23.9 Subject 10 18.9 22.6 17.3 23.8 Ct value -RT
Ct value HCT116 cell line: 10.8 30.6 Positive control No template
(no 30.8 cDNA): Negative control
Conclusions
[0160] These examples demonstrate the efficacy of the composition
of the present invention for stabilizing RNA in samples of saliva
for prolonged periods of time at room temperature. After 8 weeks,
saliva samples from 10 subjects showed no appreciable degradation
of high molecular weight RNA (as shown in FIGS. 14A and 14B) and no
significant change in the Ct value for human 18S ribosomal RNA (as
shown in Table VI).
Example 15
Extraction and Purification of Human RNA from Infants and Young
Children
[0161] In this example, it is demonstrated that RT-PCR can be used
to detect human-specific messenger RNA in RNA recovered from the
saliva of 11 infants and young children in the composition of the
present invention. Saliva sample collection was facilitated using
foam-tipped swabs (5 swabs per subject) since said subjects were
not capable of delivering 1-2 mL of saliva directly into a
collection device. A small amount of sugar was used to stimulate
the secretion of saliva from young children. Immediately following
sample collection, scissors were used to cut the foam tips of each
swab into a collection device containing the composition (2 mL) of
the present invention, comprised of 4% SDS, 50 mM LiCDTA, 250 mM
LiCl, adjusted to pH 6.8. The samples were vigorously shaken and
stored at room temperature (RT). Following storage at RT for up to
a week, the samples were heated at 50.degree. C. for 1 hour in the
presence of proteinase K. Saliva/composition was recovered from the
foam tips by low-speed centrifugation as follows. The tips were
transferred into the barrel of a 5 mL plastic syringe placed inside
a 15 mL conical tube, which was subjected to low-speed
centrifugation. The recovered liquid was pooled with the sample
remaining in the collection device. To extract RNA from saliva
samples, aliquots were heated at 90.degree. C. for 15 mM, SDS was
precipitated with potassium chloride and, after the precipitate was
removed by centrifugation, nucleic acids were precipitated from the
supernatant with 2 volumes of cold 95% ethanol.
[0162] A portion of each precipitated nucleic acid sample from 11
subjects was used as template in Reverse Transcriptase-PCR (RT-PCR)
analysis using primers specific for human 18S ribosomal RNA.
Specifically, a portion of precipitated nucleic acid sample from
each subject was treated with DNase to remove DNA prior to the
Reverse Transcriptase step. The DNA-free sample was then mixed with
random hexanucleotide primers and M-MVL reverse transcriptase to
synthesize complementary DNA (cDNA). Finally, the cDNA was diluted
10-fold and mixed with human-specific primers (human 18S-165
forward 5' gtggagcgatttgtctggtt and human 18S-165 reverse 5'
ggacatctaagggcatcacag), Taq DNA polymerase and SybrGreen for
quantitative real-time PCR analysis. The Ct (Crossing Threshold)
values, as shown in Table VII, are inversely proportional to the
amount of 18S RNA in the sample. RNA purified from a human colon
carcinoma cell line, HCT-116, served as a positive control for
RT-PCR analysis. Negative controls include reactions in which no
M-MVL RT (-RT) or no cDNA/template was added.
TABLE-US-00020 TABLE VII Real time-PCR with primers specific for
human 18S ribosomal RNA. Saliva Sample Age of subject Ct value -RT
Ct value Subject 1 3 years 11.88 28.27 Subject 2 5 years 12.88
23.27 Subject 3 25 months 14.42 26.68 Subject 4 3 years 11.46 27.53
Subject 5 4 years 6.17 23.0 Subject 6 6 years 7.74 25.99 Subject 7
8 years 5.96 22.27 Subject 8 4 years 11.7 27.02 Subject 9 5 months
10.47 28.55 Subject 10 15 months 8.52 26.01 Subject 11 7 years
14.82 24.2 HCT116 cell line: 2.83 14.17 Positive control No
template (no 21.49 cDNA): Negative control
Conclusions
[0163] These examples demonstrate the efficacy of the composition
of the present invention for stabilizing RNA in samples of saliva
collected non-invasively from infants and young children, i.e.
individuals not capable of expectorating saliva directly into a
collection device. Large amounts of human RNA, suitable for RT-PCR
analysis, can be recovered from the saliva of said
`non-spitters`.
Example 16
Protocol for Obtaining from the Nasal Cavity of Subjects a Novel
Source of Human RNA
[0164] To collect anterior nasal or nasopharyngeal samples from a
subject, a variety of implements may be used. Mucosal cells may be
scraped using rigid or flexible brushes, swabs, or plastic/wood
scrapers and cells may be flushed from the nasal cavity by
introducing a liquid (e.g., saline) and recovering the liquid. For
example, a rigid swab/brush can be placed in the anterior of the
nose and a flexible swab/brush into the posterior nasopharyngeal
cavity and used to collect mucosal secretions and to gently rub off
cells from the mucosal membrane. Samples collected with said liquid
and/or implement(s) can be delivered into a collection device
containing the composition of the present invention. In situations
where it is desirable to introduce a volume of said liquid that is
greater than the volume of the composition, a correspondingly
larger amount of composition of the present invention would be
provided. A cutting device (e.g. scissors) may be used to shorten
the length of a swab's/applicator's handle to permit closure of the
collection device. Alternately, swabs or brushes with handles that
snap under pressure, as well as swabs or brushes with a moulded
breakpoint in the handle/shaft, can be used to facilitate sample
collection. The `full length` handle facilitates the collection of
a sample and shortening of the swab/brush at the engineered break
point permits a better fit into the collection device. It is also
feasible to recover RNA from tissue samples taken from the nasal
cavity. Fresh tissue specimens/biopsies (e.g., normal nasal mucosa
or nasal polyp tissue) obtained from patients undergoing
rhinoplasty or endoscopic sinus surgery can be collected in the
composition of the present invention for subsequent RNA
isolation.
[0165] The collection of a nasal sample (anterior nasal or
nasopharyngeal) from an infant or child generally corresponds to
the procedures used for adult humans. It will be clear to the
skilled worker that the swab or implement selected to collect such
samples must 1) be appropriately sized and shaped to fit within and
reach the intended cavity (e.g. nostril, nasopharyngeal cavity), 2)
be made from materials considered safe (e.g. free of chemical
residues) and clean/sterile (e.g. nucleic acid-free), and 3) have a
handle with appropriate flexibility/rigidity and length to
facilitate sample collection. If the sample collection implement is
a swab, the end of the swab intended to collect sample (e.g. foam
mitt) should be tightly adhered to the handle.
[0166] Once the nasal secretion, scraping, and/or tissue is
collected and introduced into the collection device/container, the
sample is immediately mixed with the composition of the present
invention. The RNA-containing sample can be maintained at room
temperature for months. A portion of the RNA-containing sample in
aqueous solution can be used as a RNA template for a reverse
transcription (RT) reaction to produce complementary DNA (cDNA),
which can then be used in a PCR reaction.
Example 17
Stability of RNA in Human Nasal Samples at Room Temperature for 4
Weeks
[0167] In this example, the composition comprised 4% SDS, 50 mM
LiCDTA, 250 mM LiCl, adjusted to pH 6.8. Anterior nasal samples
were collected with 2 foam-tipped swabs per subject. Immediately
following sample collection, scissors were used to cut the foam
tips of each swab into a collection device containing the
composition (2 mL) of the present invention. The samples were
vigorously shaken and stored at room temperature (RT). Following 1
day and 4 weeks storage at RT, an aliquot from each sample was
analysed. To extract RNA from nasal samples, the aliquots were
heated at 50.degree. C. for 1 hour in the presence of proteinase K,
then at 90.degree. C. for 15 min. SDS was precipitated with
potassium chloride and, after the precipitate was removed by
centrifugation, nucleic acids were precipitated from the
supernatant with 2 volumes of cold 95% ethanol.
[0168] A portion of each precipitated nucleic acid sample from 7
subjects was used as template in Reverse Transcriptase-PCR (RT-PCR)
analysis using primers specific for human 18S ribosomal RNA.
Specifically, a portion of precipitated nucleic acid sample from
each subject was treated with DNase to remove DNA prior to the
Reverse Transcriptase step. The DNA-free sample was then mixed with
random hexanucleotide primers and M-MVL reverse transcriptase to
synthesize complementary DNA (cDNA). Finally, the cDNA was diluted
10-fold and mixed with human-specific primers (human 18S-165
forward 5' gtggagcgatttgtctggtt and human 18S-165 reverse 5'
ggacatctaagggcatcacag), Taq DNA polymerase and SybrGreen for
quantitative real-time PCR analysis. The Ct (Crossing Threshold)
values, as shown in Table VIII, are inversely proportional to the
amount of 18S RNA in the sample. RNA purified from a human colon
carcinoma cell line, HCT-116, served as a positive control for
RT-PCR analysis. Negative controls include reactions in which no
M-MVL RT (-RT) or no cDNA/template was added.
TABLE-US-00021 TABLE VIII Real time-PCR with primers specific for
human 18S ribosomal RNA. 1 day 4 week 4 week aliquot 1 day aliquot
aliquot aliquot -RT Nasal Sample Ct value -RT Ct value Ct value Ct
value Subject 1 13.04 30.62 14.81 28.65 Subject 2 15.21 31.64 18.4
29.16 Subject 3 15.63 31.51 12.89 28.08 Subject 4 11.42 28.89 12.58
27.49 Subject 5 15.26 31.83 15.69 27.67 Subject 6 14.06 30.62 13.34
28.18 Subject 7 14.68 30.04 15.42 28.46 HCT116 cell line: 4.92
35.37 4.92 27.50 Positive control No template (no 27.12 29.23
cDNA): Negative control
Conclusions
[0169] This example demonstrate the efficacy of the composition of
the present invention for stabilizing RNA in samples collected from
the nasal cavity for long periods of time at room temperature.
After 4 weeks, anterior nasal samples from 7 subjects showed no
significant change in the Ct value for human 18S ribosomal RNA (as
shown in Table VIII). Importantly, this example demonstrates that
the nasal cavity is novel source of human RNA.
Example 18
Protocol for Obtaining Saliva from a Human Subject
[0170] This example, as shown in FIG. 15, provides one example of
the steps that may be followed to collect saliva from a
subject.
[0171] Note that, on average, it can take approximately one minute
to provide a sample of saliva when sugar is used. If user has
difficulty making enough saliva, a little more sugar can be used.
It is desirable to provide the sample quickly, finishing giving the
sample within five minute. Sugar substitutes may also be used. Once
the saliva sample is collected in a composition of the present
invention, the sample may be stored at room temperature
(15-30.degree. C.)
Example 19
Protocol for the Purification of RNA
[0172] This example provides one example of the steps that may be
followed to isolate RNA from a sample stored using the composition
of the present invention. In this example, a composition of the
present invention is combined with a sample (such as saliva), and
stored at room temperature. The sample/composition mixture can be
subsequently processed to purify RNA from the sample, using the
following steps:
Reagents and Equipment
[0173] 1. Neutralizer solution. 2. Ethanol solutions: 70% and 80%
(room temperature), 95% (-20.degree. C.). 3. Qiagen RNeasy Micro
Kit (Cat. No. 74004) and instructions. Components of the RNeasy
kit: RLT buffer, MinElute spin column, collection tubes, RW1
buffer, DNase I stock solution, RDD buffer, RPE buffer and
RNase-free water. Alternatively, the Qiagen RNeasy Mini Kit (Cat.
No. 74104) can be used in combination with the Qiagen RNase-Free
DNase Set (Cat. No. 79254).
Steps Prior to Purification
[0174] 1. When samples combined with a composition of the present
invention are received (e.g., in the lab), shake very vigorously
for 8 seconds or longer. 2. Samples may be stored at room
temperature for up to 8 weeks or stored frozen at -20.degree. C.
indefinitely. 3. Prior to purification, incubate the entire sample
in the original vial at 50.degree. C. for one hour in a water bath
or for 2 hours in an air incubator.
Initial Purification
[0175] 1. Remove a 250-500 .mu.L aliquot to a 1.5 mL
microcentrifuge tube. (1000 .mu.L aliquot should be processed in 2
tubes). 2. Incubate the aliquot at 90.degree. C. for 15 minutes,
then cool to room temperature. 3. Add 1/25.sup.th volume of
Neutralizer solution e.g., 10 .mu.L Neutralizer for a 250 .mu.L
sample. Incubate on ice for 10 minutes. 4. Centrifuge at maximum
speed (>13,000.times.g) for 3 minutes. 5. Taking care not to
disturb the pellet, carefully remove supernatant to a fresh tube;
discard the pellet. 6. Add 2 volumes of cold 95% EtOH (ethanol).
Mix thoroughly by inversion, vortexing or shaking. 7. Incubate at
-20.degree. C. for 30 minutes. 8. Collect precipitate by
centrifugation at maximum speed (>13,000.times.g) for 3 minutes.
9. Carefully remove and discard the supernatant, taking care to
avoid disturbing the pellet. 10. Dissolve the pellet in 350 .mu.L
buffer RLT (RNeasy) by vigorous vortexing, taking care to ensure
that the pellet is completely dissolved. 11. Add 1 volume (350
.mu.L) of 70% ethanol. Mix well by vortexing. 12. Proceed
immediately to the Qiagen RNeasy Sample Purification
instructions.
Qiagen RNeasy Purification Procedure
[0176] Start at step #5 of the Qiagen RNeasy MicroKit "Total RNA
Isolation from Animal Cells" Protocol (noting the slight
modification to elution step #13).
5. Transfer the sample onto an RNeay MinElute spin column in a 2 mL
collection tube. Close the lid and centrifuge for 15 sec at
>8000.times.g. Discard the flow-through. Reuse the collection
tube in step 6. 6. Add 350 .mu.L of buffer RW1 to the RNeasy
MinElute spin column. Close the lid and centrifuge for 15 sec at
>8000.times.g. Discard the flow-through. Reuse the collection
tube in step 8. 7. Add 10 .mu.L DNase I stock solution to 70 .mu.L
buffer RDD. Mix by gently inverting the tube. 8. Add the DNase I
incubation mix (80 .mu.L) directly onto the RNeasy MinElute spin
column membrane and incubate on the benchtop for 15 minutes. 9. Add
350 .mu.L buffer RW1 to the RNeasy MinElute spin column. Close the
lid and centrifuge for 15 sec at >8000.times.g. Discard the
flow-through and collection tube. 10. Place the RNeasy MinElute
spin column into a fresh 2 mL collection tube. Add 500 buffer RPE
to the spin column. Close the lid and centrifuge for 15 sec at
>8000.times.g. Discard the flow-through. Reuse the collection
tube in step 11. 11. Add 500 .mu.L of 80% ethanol to the RNeasy
MinElute spin column. Close the lid and centrifuge for 2 minutes at
>8000.times.g. Discard the flow-through and collection tube. 12.
Place the RNeasy MinElute spin column into a fresh 2 mL collection
tube. Open the lid of the spin column and centrifuge at full speed
for 5 minutes. Discard the flow-through and collection tube. 13.
Place the RNeasy MinElute spin column into a fresh 1.5 mL
collection tube. Add 25 .mu.L of RNase-free water directly to the
center of the spin column membrane. Incubate at room temperature
for 5 minutes. Close the lid and centrifuge for 1 minute at full
speed to elute the RNA.
Example 20
Extraction and Purification Human RNA from Children from Nasal
Samples in the Composition of the Present Invention
[0177] In this example, RT-PCR was used to detect human-specific
messenger RNA in RNA recovered from the anterior nasal cavity of 6
young children in the composition of the present invention. Samples
were collected using two foam-tipped swabs, one swab per nostril.
Specifically, an adult inserted the foam tip into the child's
anterior/lower nasal cavity and swabbed the mucosal membrane with
the foam tip by moving the swab in a circular motion. The procedure
was repeated for the second nostril with a fresh swab. Immediately
following sample collection, scissors were used to cut the foam tip
of each swab into a collection device containing the composition (2
mL) of the present invention, comprised of 4% SDS, 50 mM LiCDTA,
250 mM LiCl, adjusted to pH 6.8. The samples were vigorously shaken
and stored at room temperature (RT).
[0178] Following storage at RT for 1-4 days, the samples were
heated at 50.degree. C. for 1 hour in the presence of proteinase K.
The nasal sample/composition mixture absorbed by the foam tips was
recovered by low-speed centrifugation as follows. The foam tips (2
per child) were transferred into the barrel of a 5 mL plastic
syringe placed inside a 15 mL conical tube, which was subjected to
low-speed centrifugation. The nasal sample/composition recovered
from the foam tips into the 15 mL conical tube was pooled with the
nasal sample/composition still in the collection device. To extract
RNA from nasal samples, aliquots (500 .mu.L) were heated at
90.degree. C. for 15 min, SDS was precipitated with potassium
chloride, the precipitate was removed by centrifugation, and
nucleic acids were precipitated from the resultant supernatant with
2 volumes of cold 95% ethanol. The final nucleic acid pellet was
dissolved in ribonuclease-free water.
[0179] A portion of each precipitated nucleic acid sample from 6
children was used as template in Reverse Transcriptase-PCR (RT-PCR)
analysis with primers specific for human 18S ribosomal RNA (18S
rRNA). Specifically, a portion of precipitated nucleic acid sample
from each subject was treated with DNase to remove DNA prior to the
Reverse Transcriptase step. The DNA-free sample was then mixed with
random hexanucleotide primers and M-MVL reverse transcriptase to
synthesize complementary DNA (cDNA). Finally, the cDNA was diluted
10-fold and mixed with 18S rRNA primers (18S-165 forward 5'
gtggagcgatttgtctggtt and 18S-165 reverse 5' ggacatctaagggcatcacag),
Taq DNA polymerase and Syto9 for quantitative real-time PCR
analysis. The Ct (Crossing Threshold) values, as shown in Table IX,
are inversely proportional to the amount of 18S rRNA in the sample.
RNA purified from a human colon carcinoma cell line, HCT-116,
served as a positive control for RT-PCR analysis. Negative controls
include reactions in which no M-MVL RT (-RT) or no cDNA/template
was added.
TABLE-US-00022 TABLE IX Real time-PCR with primers specific for 18S
ribosomal RNA. Nasal Sample Age of subject Ct value -RT Ct value
Subject 1 6 years 7.27 31.2 Subject 2 6 years 12.13 31.29 Subject 3
5 years 12.8 31.02 Subject 4 4 years 18.27 30.72 Subject 5 7 years
12.9 31.43 Subject 6 4 years 17.08 29.78 HCT116 cell line: 5.79
29.84 Positive control No template (no 29.31 cDNA): Negative
control
Conclusions
[0180] This example demonstrates the efficacy of the composition of
the present invention for extracting and stabilizing RNA in
anterior nasal samples from children. Large amounts of human RNA,
suitable for RT-PCR analysis, can be recovered from children with
this minimally invasive collection method and stabilizing
composition.
Example 22
Protocol for Obtaining Sample from the Nasal and Oral Cavities of
Livestock
[0181] Nasal and oral samples can be collected from livestock,
including beef cattle, dairy cows, sheep, goats, hogs, poultry and
horses. In a specific, non-limiting example, nasal or oral samples
can be readily collected from dairy cows (Bovidae Bos taurus) while
the cow is standing in stanchions in the milking parlour, or while
tied in pens. Depending on the age, size, and/or strength of the
animal, the assistance of a handler may, or may not, be required to
restrain/steady the animal's head for nasal sample collection or
open the animal's mouth for oral sample collection. A variety of
implements may be used to collect nasal and oral samples from
livestock. For example, implement(s) the same as or similar to
those described above in Example 16 may be used. The implement(s)
should be appropriately sized, taking into account the dimensions
of the cavity into which the implement will be inserted. It will be
clear that veterinary assistance may not be need using this
method.
Example 23
RNA Stabilized in Nasal Samples from Holstein Dairy Cows (Bovidae
bos) Using the Composition of the Present Invention
[0182] In this example, the composition comprised 4% SDS, 50 mM
LiCDTA, 250 mM LiCl, adjusted to pH 6.8. Anterior nasal samples
were collected with one large foam-tipped swab (head length 2.6 cm,
head width 1.2 cm, swab length 15.1 cm) per cow. In each case, the
cow's head was steadied by a `handler` while a `collector` quickly
inserted the foam tip of the swab into the cow's nostril,
specifically the anterior nose. The foam tip was quickly wiped
against the mucous membrane of the anterior nose/nostril and then
withdrawn. For the most part, a `handler` is not needed for the
collection of nasal samples from calves. Immediately following
collection, scissors were used to cut the foam tip of the swab into
a collection device containing the composition (2 mL) of the
present invention. The samples were vigorously shaken and stored at
room temperature (RT).
[0183] Following 16 days at RT, the sample/composition mixture
absorbed by the foam tip was recovered by low-speed centrifugation
as follows. Forceps were used to transfer the foam tip into the
barrel of a 5 mL plastic syringe situated inside a 15 mL conical
tube, which was subjected to low-speed centrifugation. The
sample/composition recovered from foam tips by this process was
pooled with the sample/composition in the collection device. To
extract RNA from nasal samples, each aliquot was heated at
50.degree. C. for 1 hour in the presence of proteinase K and then
90.degree. C. for 15 min, SDS was precipitated with potassium
chloride and, after the precipitate was removed by centrifugation,
nucleic acids were precipitated from the supernatant with 2 volumes
of cold 95% ethanol. The final nucleic acid pellet was dissolved in
ribonuclease-free water.
[0184] A portion of each precipitated nucleic acid sample from 8
dairy cows was used as template in Reverse Transcriptase-PCR
(RT-PCR) analysis using primers for 18S ribosomal RNA.
Specifically, a portion of precipitated nucleic acid sample from
each dairy cow was treated with DNase to remove DNA prior to the
Reverse Transcriptase step. The DNA-free sample was then mixed with
random hexanucleotide primers and M-MVL reverse transcriptase to
synthesize complementary DNA (cDNA). Finally, the cDNA was diluted
10-fold and mixed with 18S ribosomal RNA primers (18S-165 forward
5' gtggagcgatttgtctggtt and 18S-165 reverse 5'
ggacatctaagggcatcacag), Taq DNA polymerase and Syto9 for
quantitative real-time PCR analysis. The Ct (Crossing Threshold)
values, as shown in Table X, are inversely proportional to the
amount of 18S ribosomal RNA in the sample. HCT-116 RNA served as a
positive control for RT-PCR analysis. Negative controls include
reactions in which no M-MVL RT (-RT) or no cDNA/template was
added.
TABLE-US-00023 TABLE X Nasal Sample Ct value -RT Ct value Cow 1
15.31 31.75 Cow 2 13.94 32.17 Cow 3 19.02 30.64 Cow 4 13.1 28.38
Cow 5 14.14 31.31 Cow 6 16.69 31.4 Cow 7 18.68 31.03 Cow 8 11.49
31.83 HCT116 cell line: 5.95 27.07 Positive control No template (no
31.53 cDNA): Negative control
Conclusions
[0185] This example demonstrates the efficacy of the composition of
the present invention for extracting and stabilizing RNA in samples
collected from the anterior nasal cavity of dairy cows (as shown in
Table X). Importantly, this example demonstrates that the nasal
cavity is a novel and abundant source of messenger RNA in
cattle.
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[0199] All publications, patents and patent applications mentioned
in this Specification are indicative of the level of skill of those
skilled in the art to which this invention pertains and are herein
incorporated by reference to the same extent as if each individual
publication, patent, or patent applications was specifically and
individually indicated to be incorporated by reference.
[0200] The invention being thus described, it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the invention,
and all such modifications as would be obvious to one skilled in
the art are intended to be included within the scope of the
following claims.
* * * * *