U.S. patent application number 13/446444 was filed with the patent office on 2013-03-14 for vessel for blood sampling.
This patent application is currently assigned to PreAnalytiX GmbH. The applicant listed for this patent is Elke Helftenbein. Invention is credited to Elke Helftenbein.
Application Number | 20130066234 13/446444 |
Document ID | / |
Family ID | 7877311 |
Filed Date | 2013-03-14 |
United States Patent
Application |
20130066234 |
Kind Code |
A1 |
Helftenbein; Elke |
March 14, 2013 |
VESSEL FOR BLOOD SAMPLING
Abstract
The present invention relates to a vessel for withdrawing blood,
the vessel containing a solution which comprises a guanidinium
salt, a buffer substance, a reducing agent, and/or a detergent as
components. The vessel is particularly suited for withdrawing blood
which is to be analyzed with respect to nucleic acids.
Inventors: |
Helftenbein; Elke;
(Stuttgart, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Helftenbein; Elke |
Stuttgart |
|
DE |
|
|
Assignee: |
PreAnalytiX GmbH
Hombrechtikon
CH
|
Family ID: |
7877311 |
Appl. No.: |
13/446444 |
Filed: |
April 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11506164 |
Aug 17, 2006 |
RE43389 |
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13446444 |
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09762643 |
May 18, 2001 |
6776959 |
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PCT/EP99/05857 |
Aug 12, 1999 |
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11506164 |
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Current U.S.
Class: |
600/577 ;
422/547; 536/25.4 |
Current CPC
Class: |
Y10T 436/25 20150115;
A61B 5/150351 20130101; A61B 5/150755 20130101; C12N 15/1003
20130101; C12Q 1/6806 20130101; A61B 5/1438 20130101; A61B 5/15003
20130101; A61B 5/154 20130101; Y10T 436/2525 20150115 |
Class at
Publication: |
600/577 ;
422/547; 536/25.4 |
International
Class: |
A61B 5/15 20060101
A61B005/15 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 12, 1998 |
DE |
19836559.4 |
Claims
1-160. (canceled)
161. A blood withdrawing vessel comprising an evacuated chamber
which is provided for receiving withdrawn blood containing a
nucleic acid-stabilizing aqueous solution for stabilizing nucleic
acids for quantitative analysis in withdrawn blood directly upon
contact with the solution, wherein the solution comprises the
following components: a guanidinium salt in a concentration of 1 to
8.0 M; a buffer substance in a concentration of 10 to 300 mM; and a
detergent.
162. The vessel according to claim 161, characterized in that the
guanidinium salt is selected from guanidinium thiocyanate and
guanidinium chloride.
163. The vessel according to claim 161, characterized in that the
guanidinium salt is present in a concentration of 2.5 to 8.0 M.
164. The vessel according to claim 161, characterized in that the
buffer substance is selected from Tris, HEPES, MOPS, citrate and
phosphate buffer.
165. The vessel according to claim 161, characterized in that the
detergent is selected from Triton-X-100, NP-40, polydocanol and
Tween 20.
166. The vessel according to claim 161, characterized in that the
pH of the solution is between 4.0 and 7.5.
167. The vessel according to claim 161, characterized in that it
contains withdrawn blood.
168. A method of withdrawing blood, comprising the step of directly
introducing the blood into a vessel according to claim 161.
169. The method according to claim 168, characterized in that an
amount of blood is withdrawn that is 0.1 to 4 times the volume of
the solution in the vessel.
170. The method according to claim 169, characterized in that the
concentration of the guanidinium salt after the blood is introduced
is between 1.0 M and 5 M.
171. A method for quantitatively stabilizing nucleic acids from
blood, comprising the step of introducing blood into a vessel
according to claim 161 and, optionally, isolating the nucleic acids
with conventional methods.
172. The method according to claim 170, characterized in that the
pH of the resultant mixture of the solution and blood is between
5.0 and 7.6.
173. The method according to claim 172, characterized in that the
concentration of the guanidinium salt, after blood is introduced,
is between 1.5 and 5 M.
174. A vessel comprising an interior, the interior comprising an
aqueous solution that comprises a guanidinium salt, a buffer and a
detergent, wherein the interior is evacuated, and wherein the
aqueous solution is capable of lysing blood cells and stabilizing
nucleic acids for quantitative analysis.
175. The vessel of claim 174, wherein the vessel comprises a tube
having an open end sealed by a septum.
176. The vessel of claim 174, wherein the evacuation is effective
for drawing a specific volume of a fluid sample into the
interior.
177. The vessel of claim 174, wherein the guanidinium salt, the
buffer and the detergent are present in amounts effective to
provide cell lysis and stabilization of nucleic acids in a fluid
sample by inactivation of nucleases.
178. The vessel of claim 177, wherein the buffer is effective, upon
mixing with the specific volume of a blood sample, to provide the
resultant mixture with a pH of 5.0 to 7.6.
179. The vessel of claim 178, wherein the buffer is effective to
provide the resultant mixture with a pH of 6.3 to 6.9.
180. The vessel of claim 174, wherein the pH of the aqueous
solution in the vessel is 4.0 to 7.5.
181. The vessel of claim 180, wherein the pH is 5 to 6.
182. The vessel of claim 176, wherein the specific volume is about
0.1 to about 4 times the volume of the solution.
183. The vessel of claim 174, wherein the guanidinium salt is one
or more salts selected from the group consisting of guanidinium
thiocyanate and guanidinium chloride.
184. The vessel of claim 174, wherein the buffer is selected from
the group consisting of: Tris (tris(hydroxymethyl)aminomethane),
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS
(4-morpholinepropanesulfonic acid), MES (4-morpholineethanesufonic
acid), citrate and phosphate buffer.
185. The vessel of claim 174, wherein the detergent is selected
from the group consisting of: (polyethylene glycol tert-octylphenyl
ether), (polyethylene glycol 4-nonylphenyl ether), Polydocanol
(dodecyl polyethylene glycol ether) and (polyethylene glycol
sorbitan monolaurate).
186. The vessel of claim 174, wherein the solution comprises a
single buffer and a single detergent.
187. The vessel of claim 174, wherein the aqueous solution consists
essentially of the guanidinium salt, the buffer and the
detergent.
188. A process for collecting and stabilizing a blood sample for
quantitative nucleic acid analysis, the process comprising:
providing a vessel comprising an interior, the interior comprising
an aqueous solution that comprises a guanidinium salt, a buffer and
a detergent; and drawing the blood sample from a blood vessel
directly into the interior, wherein the aqueous solution causes
lysis of cells in the blood and stabilizes nucleic acids in the
sample.
189. The process of claim 188, wherein the interior is evacuated
and wherein the evacuation is used to draw the blood sample.
190. The process of claim 188, wherein the vessel comprises a tube
having an open end sealed by a septum.
191. The process of claim 188, wherein the evacuation is effective
for drawing a specific volume of the blood into the interior.
192. The process of claim 188, wherein the guanidinium salt, the
buffer and the detergent are present in amounts effective to
provide cell lysis and stabilization of nucleic acids in the sample
by inactivation of nucleases.
193. The process of claim 188, wherein the buffer is effective,
upon drawing the blood into the interior, to provide the resultant
mixture with a pH of 5.0 to 7.6.
194. The process of claim 193, wherein the buffer is effective,
upon drawing the blood into the interior, to provide the resultant
mixture with a pH of 6.3 to 6.9.
195. The process of claim 191, wherein the specific volume is about
0.1 to about 4 times the volume of the solution.
196. The process of claim 188, wherein the guanidinium salt is one
or more salts selected from the group consisting of guanidinium
thiocyanate and guanidinium chloride.
197. The process of claim 188, wherein the buffer is selected from
the group consisting of: Tris (tris(hydroxymethyl)aminomethane),
HEPES (4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid), MOPS
(4-morpholinepropanesulfonic acid), MES (4-morpholineethanesufonic
acid), citrate and phosphate buffer.
198. The process of claim 188, wherein the detergent is selected
from the group consisting of: (polyethylene glycol tert-octylphenyl
ether), (polyethylene glycol 4-nonylphenyl ether), Polydocanol
(dodecyl polyethylene glycol ether) and (polyethylene glycol
sorbitan monolaurate).
199. The process of claim 188, wherein the pH of the aqueous
solution in the vessel prior to the drawing step is 4.0 to 7.5.
200. The process of claim 199, wherein the pH of the aqueous
solution in the vessel prior to the drawing step is 5 to 6.
201. The process of claim 188, further comprising the step of
engaging the vessel with a blood sampling accessory.
202. The process of claim 188, wherein the solution comprises a
single buffer and a single detergent.
203. The process of claim 188, wherein the aqueous solution
consists essentially of the guanidinium salt, the buffer and the
detergent.
204. The process of claim 188, further comprising the step of
isolating the nucleic acids after the drawing step.
205. The process of claim 204, wherein the isolating step is
performed at least 3 days after the drawing step.
206. The process of claim 205, wherein the isolating step is
performed at least 6 days after the drawing step.
207. The process of claim 206, wherein the isolating step is
performed at least 8 days after the drawing step.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Reissue patent
application Ser. No. 11/506,164 filed Aug. 17, 2006 which claims
priority to application Ser. No. 09/762,643, filed May 18, 2001
(now U.S. Pat. No. 6,776,959, issued Aug. 17, 2004). Applicant also
claims priority under 35 U.S.C. .sctn.119 of GERMAN Application No.
198 36 559.4 filed on Aug. 12, 1998. Applicant also claims priority
under 35 U.S.C. .sctn.120 of PCT/EP99/05857, filed on Aug. 12,
1999. The international application under PCT article 21(2) was not
published in English.
[0002] The present invention relates to a vessel for withdrawing
blood, and the blood withdrawn should especially be used for
stabilizing and analyzing nucleic acids.
[0003] When blood is taken, it is normally collected in vessels
which already contain anticoagulants such as heparin, citrate or
EDTA. The blood is thereby prevented from coagulating. The blood
samples obtained thereby can be stored at suitable temperatures for
a long time. This way of obtaining blood has, however, considerable
drawbacks when nucleic acids such as (m)RNA or DNA are to be
analyzed. For such purposes the nucleic acids contained in the
sample should optimally be stabilized already at the moment of
withdrawal, i.e. a degradation of the existing nucleic acids should
be prevented, but also the new synthesis of mRNA.
[0004] This objective of a stable storage of the nucleic acids
contained in the sample material, i.e. from the moment of
withdrawal, has not been achieved yet in practice for the following
reasons:
[0005] Cells contain nucleases, enzymes, which destroy nucleic
acids as soon as they come into contact with the substrates thereof
(RNA, DNA). The effect of cellular and extracellular nucleases is
normally under physiological control as long as the cells are in
their normal environment. The withdrawal of blood effects more or
less strong changes in the nucleic acids contained in the cells.
Nucleases are then released within the cells and/or by the lysis of
cells to the outside. Moreover, nucleic acids are synthetized more
or less strongly. In particular the long-term storage of blood
leads to aging and destruction of the cells.
[0006] Another problem arising in the long-term storage of blood
samples obtained according to standard withdrawal methods is the
considerable change in the sample material. Such changes, e.g.
strong lysis of cells, may have the effect that the standard
methods for isolating nucleic acids no longer function in an
adequately efficient way.
[0007] Apart from the problems regarding a stable storage of
nucleic acids contained in the sample material, further
difficulties arise in the conventional method for withdrawing
blood. The conventional anticoagulants are often not separated
efficiently enough during isolation of nucleic acids and interfere
with in the subsequent analysis of nucleic acids, e.g. in the case
of amplification by means of PCR (polymerase chain reaction).
Heparin is e.g. a generally known inhibitor of PCR.
[0008] Finally, the question arises in the quantitative analysis of
nucleic acids how the whole method ranging from sampling to the
measurement of nucleic acids can be controlled under standardized
conditions. Ideally, a quantitatively and qualitatively defined
standard nucleic acid should already be added to the sample
material during withdrawal and should be subjected to the whole
process of sampling and determination. This can also not be
accomplished with the conventional withdrawal systems.
[0009] A further drawback of conventional blood withdrawal is the
risk of transferring infectious material because manual process
steps have so far been needed for the isolation of nucleic acids.
Contact with potentially infectious germs cannot be ruled out.
[0010] In the literature there is described a method in which the
blood sample is mixed with guanidinium salt directly after
withdrawal from a patient (EP 0 818 542 A1). In this method the
guanidinium salt is present in powder form to thereby exploit the
increased stability of the guanidinium salt. This method, however,
has serious drawbacks because the salt, for instance, must first
dissolve in the added blood. This dissolution process depends, in
particular, on the temperature and cannot be controlled because of
the nontransparent sample material used. The use of a corresponding
product for diagnostic medical purposes is thus very
problematic.
[0011] Furthermore, nucleases are extremely active enzymes which
can only be inhibited under extremely denaturing conditions.
Denaturation depends on the concentration of the guanidinium salt
in solution. An inhibiting concentration of guanidinium salt in
solution does not exist in the cited method right from the
beginning. Thus, there is an uncontrolled degradation of nucleic
acids during the dissolution process. Moreover, in this method the
addition of reducing agents is omitted, without which an efficient
inhibition, in particular of RNases, is not ensured (see Example
no. 5).
[0012] Moreover, the sample prepared in this way cannot directly be
used for the further nucleic acid isolation on glass surfaces.
Moreover, the use of guanidinium salt powder does not permit the
addition of internal nucleic acid standards. Such standards are
mandatory for process control and exact quantification.
[0013] The present invention has been based on the technical
problem of providing a vessel for withdrawing blood which does not
have the drawbacks of the prior art. In particular, it should be
possible to subject the sample taken with the vessel directly to
the standard methods for analyzing nucleic acids without the need
for further sample preparation steps.
[0014] According to the invention this problem is solved by a
vessel for withdrawing blood, the vessel containing an aqueous
solution comprising the following components:
[0015] a guanidinium salt;
[0016] a buffer substance;
[0017] a reducing agent; and/or
[0018] a detergent.
[0019] The vessel of the invention has the following advantages: 1.
Blood is already lysed at the moment of withdrawal in that the
withdrawal vessel already contains a nucleic acid-stabilizing
substance in solution. 2. The nucleic acid-stabilizing substance is
composed such that the sample material, in particular the nucleic
acids contained therein, are directly stabilized upon contact with
the solution. 3. In contrast to all of the former standard
withdrawal systems, such as EDTA or heparin-containing withdrawal
vessels, the stabilized sample need no longer be handled as
infectious material. 4. The nucleic acid-stabilizing substance is
composed such that the sample material can directly be used in
subsequent isolating methods. 5. The nucleic acid-stabilizing
substance can be separated during subsequent isolation so
efficiently that an inhibition of PCR is not observed. 6. The
nucleic acid-stabilizing substance may have added thereto an
internal standard. This permits the control of the whole method
from the moment of sampling up to the detection of nucleic
acids.
[0020] The withdrawal vessel mentioned under item 1 is a
conventional blood withdrawing vessel (small tube) which has
introduced thereinto a defined volume of a nucleic acid-stabilizing
substance. The small tube is then preferably subjected to a defined
vacuum which guarantees that only a specific volume of blood can
flow thereinto during withdrawal. The small tube can be handled by
conventional blood-taking methods. The solution contained in the
tube contains the following reagents in a specially preferred
embodiment: Guanidinium thiocyanate, Triton-X-100, dithiothreitol
and a suitable buffer system, such as citrate, Tris or HEPES. In
the described composition the solution is compatible with the
vacuum tube. This solution can be stored in the vacuum tube without
any problems and without any impairment of the desired stabilizing
function. The whole system presents no problems, in particular to
blood donors, and is safe during sampling.
[0021] The solution containing the guanidinium salt, the buffer
substance, the reducing agent and/or the detergent is stable in
storage and converts the supplied and freshly taken blood into a
material which is also stable in storage and can directly be
subjected to the standard nucleic-acid analysis kits (e.g. those of
Roche or Qiagen).
[0022] Guanidinium thiocyanate and/or guanidinium chloride are
preferred as guanidinium salt.
[0023] Preferably, the guanidinium salt is present in a
concentration of 2.0 to 8.0 M. Tris or citrate is preferred as the
buffer substance, the exact pH being preferably adjusted with HCl.
Further possible buffers are however HEPES, MOPS, citrate and
phosphate buffer, such as PBS.
[0024] The buffer concentration is preferably between 10 and 300
mM, particularly preferably between 10 and 100 mM.
[0025] Triton-X-100 is preferred as the detergent. Further possible
detergents are NP40, Tween 20, polydocanol or other detergents.
[0026] The detergent concentration is preferably at 5 to 30% (w/v),
particularly preferably at 10 to 20% (w/v).
[0027] DTT is preferred as the reducing agent, but
.beta.-mercaptoethanol, TCEP (Tris(2-carboxyethyl)phosphine) or
other reducing agents can also be used.
[0028] The preferred concentration of the reducing agent is at 0.1
to 10% (w/v), particularly preferred are 0.5 to 2% (w/v).
[0029] The pH of the solution is preferably at 3.0 to 9.0,
particularly preferably at 4.0 to 7.5, particularly preferably at 5
to 6.
[0030] The pH of the solution is in particular chosen such that a
pH ranging from 5.0 to 7.6 is set after addition of the sample
material. Particularly preferred is a pH between 6.3 and 6.9 (see
Example no. 8).
[0031] A particularly preferred solution preferably contains 4 M
guanidinium thiocyanate, 45 mM Tris/HCl, 18%, preferably 15% (w/v)
Triton-X-100, 0.8% (w/v) DTT and has a pH of 6.0.
[0032] In a further preferred embodiment the volume for receiving
the blood sample has a negative pressure which can be adjusted such
that a previously determined blood volume is sucked into the vessel
after a blood vessel has been pierced. Correspondingly evacuated
vessels are available on the market.
[0033] The vessel which contains the blood taken can then
immediately be subjected to further analyses or, however, may be
stored for a long period of time (up to several days) without any
disadvantages for the quality of the sample.
[0034] In the method of the invention the freshly taken blood is
directly contacted in the blood withdrawing vessel with the
above-described solution so that all processes which might change
the nucleic acid pattern of the sample are immediately stopped.
Therefore, the data determined at a later time with respect to the
detected nucleic acids very accurately represent the actual state
at the time of blood withdrawal, i.e. both with respect to the
quantities and the types of nucleic acids.
[0035] Preferably, the blood amount taken is 0.1 to 4 times the
solution fed into the vessel. The solution is preferably 0.5 to 5.0
ml. Thus the final concentration of guanidinium salt after blood
addition is at 1.0 to 5 M, preferably at 1 to 3 M, particularly
preferred are 2-3 M (see Example 7).
[0036] The vessel according to the invention is preferably used for
blood withdrawal when the blood sample is to be used for analyzing
nucleic acids.
[0037] The use of the above-mentioned solution as a component of
the described withdrawal system solely guarantees the immediate
lysis of the cells and the simultaneous stabilization of the sample
by immediate inactivation of the nucleases. Surprisingly, the blood
sample obtained thereby can be stored even at room temperature or
higher for several days. Moreover, the withdrawal system guarantees
a contamination-free and non-infectious handling ranging from
sampling via nucleic acid isolation to analysis. In the
conventional methods of nucleic acid isolation, additional handling
steps have so far been required (e.g. the transfer of the blood
sample taken into the reagents for nucleic acid isolation, etc.),
which entails an additional risk of infection.
[0038] The sample obtained with the blood withdrawing system is
compatible with all of the conventional standard methods of nucleic
acid isolation. Particular attention should here be paid to methods
which are based on the binding of nucleic acids to glass surfaces,
but also sequence-specific binding to complementary nucleic acid
and solvent-based extraction methods.
[0039] Thus the invention as described consists of a blood
withdrawing system which is conceived such that the following
conditions are satisfied. 1. Controlled sampling and simultaneous
stabilization of the nucleic acids (DNA, RNA) contained in the
sample material. 2. Sampling in which the use of anticoagulants can
be completely omitted. 3. The sample obtained by way of the
above-described blood withdrawing system can be used in a universal
manner in all of the known systems for isolating nucleic acids. 4.
The blood withdrawing system is stable in storage.
[0040] Additionally, it has surprisingly been found that the sample
obtained by way of the described withdrawal system can be stored in
the vessel for a long period of time without degradation of the
nucleic acids (see Examples 2, 3, 7, 8).
[0041] The following examples will explain the invention:
EXAMPLE 1
[0042] The blood withdrawing system may be composed in a preferred
embodiment as follows (see FIG. 1): A small tube is filled with a
defined volume of the nucleic acid-stabilizing substance and is
provided with a defined vacuum and sealed with a septum. The septum
is constructed such that it is compatible with the standard
sampling accessories (cannula, etc.). In the present example 2.2 ml
reagent was supplied and the vacuum was adjusted such that exactly
2.2 ml blood could flow in during sampling. The nucleic acids
contained in the inflowing blood flow were immediately converted
into a stable form.
[0043] General preliminary remark regarding the following
examples.
[0044] In all of the examples described hereinbelow, the nucleic
acid-stabilizing substance (N-sS) had, unless indicated otherwise,
the following composition: 45 mM Tris, 5 M guanidinium thiocyanate
(GTC), 0.8% (w/v) dithiothreitol (DTT), 18% (w/v) Triton-X-100, pH
6.0.
[0045] In all of the examples described, the nucleic
acid-stabilizing substance was, unless indicated otherwise, mixed
with the sample in the ratio of 1 to 1 (1 volume N-sS plus 1 volume
sample material). A lower concentration of N-sS, e.g. 1 volume N-sS
plus 5 volumes sample, might effect a degradation of RNA.
[0046] Blood was stabilized for all examples by directly feeding
the blood upon withdrawal into the small tube mixed with N-sS.
EXAMPLE 2
[0047] Stability of nucleic acid after mixture of sample material
and N-sS. Isolation of RNA and DNA from the sample lysate with
silica-derivatized surfaces.
[0048] Material and Method
[0049] The sample material for the DNA and RNA isolation was
directly used after withdrawal, after storage at 4.degree. C. for 6
days, and after storage at -20.degree. C. for 1 month.
[0050] The HighPure RNA Isolation Kit (Boehringer Mannheim, cat.
no. 1828 665) was used for isolating RNA (FIG. 2). The instructions
given in the package leaflet were modified as follows: A volume of
2.4 ml sample lysate was applied in 4 aliquots at 600 .mu.l each to
the column, so that a sample material of 2.4 ml lysate was applied
on the whole. All of the other steps were carried out in accordance
with the package leaflet. The RNA was finally eluted with 100 .mu.l
elution buffer.
[0051] For the isolation of DNA (FIG. 3) the QiaAmp Blood Kit
(Qiagen cat. no. 29104) was used. The standard procedure described
in the package leaflet was modified in various points: 400 .mu.l
sample volume was directly applied to the column; the binding
reagent contained in the kit was not used. 25 .mu.l proteinase-K
batch solution was added and the sample was incubated at room
temperature for 10 min. Subsequently, the column was put into a
collection vessel and centrifuged as described in the package
leaflet. All of the further steps were carried out in accordance
with the description in the package leaflet, except for the use of
ethanol. The elution volume was 200 .mu.l.
EXAMPLE 3
[0052] Isolation of mRNA from sample lysate using
streptavidin-coated magnetic particles and biotin-labeled Oligo(dT)
(FIG. 4):
[0053] Material and Method
[0054] 20 ml sample lysate was fed into a vessel. The mRNA was
isolated according to the following method: First of all 30 ml
hybridization buffer (20 mM Tris-HCl, 300 mM NaCl, 6 nM
biotin-labeled Oligo(dT), pH 7.4) was added to the lysate. 3 mg
streptavidin magnetic particles (Boehringer Mannheim) were then
added. The sample was mixed and incubated at room temperature for 5
min. The magnetic particles were separated with the help of a
magnet; the supernatant was discarded. The particles were then
resuspended in wash buffer 1 (10 mM Tris-HCl, 200 mM NaCl, 1%
Triton-X-100, pH 7.5) and washed three times with wash buffer 2 (10
mM Tris-HCl, 200 mM NaCl, pH 7.5) (wash steps: resuspension,
magnetic separation, removal of the supernatant). After the last
wash step the supernatant was completely removed and the particles
were resuspended in 20 .mu.l distilled water. The sample was heated
to 70.degree. C. for 5 min. The magnetic particles were separated,
and the supernatant which contained the mRNA was analyzed by means
of gel electrophoresis.
EXAMPLE 4
[0055] Isolation of DNA and RNA using a modified rule according to
Chomczynski and Sacchi (Analytical Biochemistry 162, 156-159
(1987)) (example of a method based on solvent extraction) (FIG.
5):
[0056] Material and Method
[0057] 2 ml sample volume was transferred from the blood
withdrawing vessel into a small tube. 0.2 ml of a 2 M sodium
acetate solution, pH 4, 2 ml phenol (water saturated) and 0.4 ml of
a chloroform-isoamyl alcohol mixture (49:1) were then added, the
sample being thoroughly mixed after addition of each solution. The
complete solution was vigorously shaken for 10 seconds and
incubated on ice for 15 minutes. The sample was centrifuged for 20
minutes at 4.degree. C. at 10000 g. After centrifugation the RNA
was in the aqueous phase; the DNA and proteins in the intermediate
and phenol phase. The aqueous phase was transferred into a new
vessel and mixed with 1 ml isopropanol. For precipitating the RNA
the sample was stored at -20 C. for 1 hour. After renewed
centrifugation at 4.degree. C. at 10000 g the RNA was pelleted. The
pellet was resuspended in 0.3 ml buffer (4 M guanidinium
thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1M
2-mercaptoisopropanol), transferred into a new 1.5 ml Eppendorf
vessel and mixed with 1 volume of isopropanol. After incubation at
-20.degree. C. for 1 hour the solution was centrifuged in an
Eppendorf centrifuge at 4.degree. C. for 10 minutes. The RNA pellet
was received in 75% ethanol and concentrated by centrifugation
(Speed vac) and dried. For further processing the sample was
dissolved in 100 .mu.l 10 mM Tris-HCl, pH 6.5.
EXAMPLE 5
[0058] Importance of reducing reagents (such as DTT) in the
stabilizing solution for the longterm stability of RNA
[0059] Material and Method
[0060] Stabilizing solution used: 4.0 M GTC; 13.5% Triton X100; 45
mM Tris//HCl; with or without 120 mM DTT. 700 .mu.l serum was mixed
with 700 .mu.l stabilizing solution. After incubation for 2 min 20
.mu.l MS2-RNA (0.8 .mu.g/.mu.l of Roche) was added. The samples
were incubated at 40.degree. C. for 180 min and then processed in
aliquots of 400 .mu.l each with the High Pure total RNA Kit of
Roche. The samples were applied in one step to the column without
addition of the binding reagent of the kit and centrifuged in
accordance with the instructions. The following wash steps and the
elution of the RNA in 50 .mu.l elution buffer were carried out in
accordance with the instructions.
[0061] The analysis was carried out by means of agarose gel (see
FIG. 6). Result: Without the addition of reducing reagents to the
stabilizing solution no long-term stabilization of RNA can be
achieved.
EXAMPLE 6
[0062] Stability of Free MS2-RNA in Serum. Kinetics of the RNA
Degradation by Sample Components
[0063] Material and Method
[0064] 250 .mu.l serum was spiked with 10 .mu.l MS2-RNA (0.8
.mu.g/.mu.l of Roche) and incubated at room temperature.
Immediately after the addition of RNA, after 2 min to 50 min, the
natural RNA degradation in serum was stopped by adding 250 .mu.l
stabilizing solution. All batches were analyzed twice. As a
standard, a sample was only mixed with MS2-RNA after addition of
the stabilizing solution to the serum and was processed in
parallel.
[0065] All samples were processed in parallel with the High Pure
viral RNA Kit of Roche. The samples were applied to the column in
one step without addition of the binding reagent of the kit and
centrifuged according to instructions. The following wash steps and
the elution of RNA in 50 .mu.l elution buffer were carried out
according to instructions. 20 .mu.l of the eluate was separated by
means of a 1.2% native agarose gel and analyzed (see FIG. 7).
[0066] Result: MS2-RNA is not stable in serum. Already 2 minutes
after addition of RNA to the serum the RNA is completely degraded.
By the addition of stabilizing solution to the serum in the ratio
of 1:1, this process can be stopped immediately, and a
stabilization of the RNA can be achieved at the time when the
stabilizing solution is added (=blood withdrawal).
EXAMPLE 7
[0067] Stability of MS2-RNA in Serum/stabilization Solution.
Dependence on the GTC Concentration
[0068] Material and Method
[0069] Stabilization solutions used: 3-5 M GTC; 13.5% Triton X100;
50 mM DTT; 42 mM Tris/HCl
[0070] pH of the solutions: about 4.0
[0071] pH of the solutions after addition of serum: about 6.7.
[0072] 2 ml serum was mixed with 2.5 ml of the respective
stabilization solutions. After an incubation time of 2 to 5 min 90
.mu.l MS2-RNA (0.8 .mu.g/.mu.l of Roche) was added and incubated at
40.degree.C. 400 .mu.l samples were taken at regular intervals and
processed with the High Pure total RNA Kit of Roche according to
Example 5. The samples were eluted in 50 .mu.l and frozen at
-20.degree. C. For the analysis of the RNA integrity 20 .mu.l of
the eluate was applied to a 1.5% agarose gel (see FIG. 8).
[0073] For the PCR analysis of the samples 10 .mu.l of the eluate
was reversely transcribed by means of AMV-RT (Roche) and
subsequently analyzed by means of PCR on the Lightcycler:
TABLE-US-00001 Batch for RT: 4.0 .mu.l AMV-RT buffer (42.degree. C.
for 1 h) 2.0 .mu.l dNTP's (final concentration 10 mM) 0.5 .mu.l
RNase inhibitor (Roche, 20 units) 1.0 .mu.l Primer 2827 (final
concentration 1 .mu.M) 1.9 .mu.l DMPC water 0.6 .mu.l AMV-RT
(Roche, 15 units) 10 .mu.l template RNA .SIGMA. 20 .mu.l
[0074] The PCR was carried out on the Lightcycler at an annealing
temperature of 61.degree. C. using SYBR-Green as detection system.
Batch for PCR:
TABLE-US-00002 1.6 .mu.l MgCl.sub.2 (batch solution 25 mM) 5.9
.mu.l DMPC water 0.25 .mu.l Primer 2827 (batch solution 20 mM) 0.25
.mu.l Primer 2335 (batch solution 20 mM) 1.0 .mu.l
SYBR-Green-Mastermix (Roche) 1.0 .mu.l RT batch (1:50 diluted)
.SIGMA. 10 .mu.l
[0075] The amplificate of the PCR was completely applied to a 2%
agarose gel (see FIG. 9).
[0076] Result:
[0077] RNA integrity at 40.degree. C. after 3 days.
[0078] The agarose gel in FIG. 8 shows 20 .mu.l of the eluted
MS2-RNA after incubation at 40.degree. C. for 3 days. After this
period distinct differences in the RNA integrity can be made out in
dependence upon the GTC content. Thus a salt content of less than 2
M in the serum/stabilization solution is of advantage to the
integrity of the RNA.
[0079] Amplificability of the RNA at 40.degree. C. after 8
days.
[0080] Although a beginning degradation of the RNA was already
detected at 40.degree. C. after 3 days, all of the RNA samples
could be amplified after an incubation of 8 days at 40.degree. C.
and clearly detected.
[0081] The amplificate of the PCR was fully applied to a 2% agarose
gel (see FIG. 9).
EXAMPLE 8
[0082] Stability of MS2-RNA in Serum/stabilization Solution:
Dependence on the pH of the
Sample Mixed with Stabilization Solution
[0083] Material and Method
TABLE-US-00003 Solution used: 4M (5M) GTC 14.4% Triton X 100 50 mM
DTT 45 mM Tris/HCl
[0084] pH after serum addition between 6.7 and 8.0
[0085] 2.5 ml stabilization solution was mixed with 2.0 ml serum.
After addition of 90 .mu.l MS2-RNA (0.8 .mu.g/.mu.l, Roche) the
samples were incubated at room temperature. The RNA was processed
at regular intervals from 500 .mu.l sample with the Roche viral RNA
kit according to Example 6 and isolated in 50 .mu.l elution buffer.
20 .mu.l of the eluate was analyzed by means of agarose gel (see
FIG. 10).
[0086] Result:
[0087] The pH of the serum/stabilization solution and thus the pH
and the buffer range of the stabilization solution are decisive for
the long-term stabilization of RNA. While at a pH of 8.0 an intact
RNA could no longer be detected already after 2 days, intact RNA is
still detectable within a pH range between 6.6 and 7.0 after 13
days of incubation at room temperature.
[0088] Apart from the pH, however, an optimally adjusted GTC
concentration is also of importance to the long-term stabilization
of RNA (see also Example 7). The illustrated example demonstrates
that a GTC final concentration in the stabilized sample of 2.2 M
GTC is better than 2.78 for a long-term stabilization of RNA.
LEGENDS
[0089] FIG. 1:
[0090] Sampling vessel with N-sS, defined vacuum, sealed with
septum.
[0091] FIG. 2:
[0092] Gel analysis (1% agarose) of RNA which was stored in the
sampling vessel for different periods of time. Column 1: Isolation
directly after sampling (no storage), column 2: storage for one
month at -20.degree. C., column 3: storage for 6 days at 4.degree.
C. The amount of the applied RNA corresponded to a blood volume of
120 .mu.l.
[0093] FIG. 3:
[0094] Gel analysis (1% agarose) of DNA which was stored in the
sampling vessel for different periods of time. Column 1: isolation
directly after sampling (no storage), column 2: storage for one
month at -20.degree. C., column 3: storage for 6 days at 4.degree.
C. The amount of the applied DNA corresponded to a blood volume of
10 .mu.l.
[0095] FIG. 4:
[0096] Gel analysis (1% agarose) of mRNA which was isolated from 10
ml blood (column 2). Molecular weight marker (column 1). In
addition to the mRNA, the rRNA bands are visible. The sharp
contours of the bands demonstrate the integrity of the nucleic
acids.
[0097] FIG. 5:
[0098] Gel analysis (1% agarose) of the RNA which was isolated from
120 .mu.l blood.
[0099] FIG. 6:
[0100] Gel analysis of isolated MS2-RNA after incubation in
serum/stabilization solution with/without DTT for 180 min at
40.degree. C.
[0101] Column 1: positive control: MS2-RNA, column 2: DNA marker,
column 3,4,5: MS2-RNA after incubation with DTT-containing
stabilization solution, column 6,7,8: MS2-RNA after incubation with
stabilization solution without DTT.
[0102] FIG. 7:
[0103] Gel analysis of isolated MS2-RNA after incubation in serum
for 0-50 min
[0104] Column 10,17: MS2-RNA standard, column 9,16: DNA marker,
column 7,8: incubation for 0 min, column 5,6: incubation for 2 min,
column 3,4: incubation for 5 min, column 1,2: incubation for 10
min, column 11,12: incubation for 15 min, column 13,14: incubation
for 30 min, column 15: incubation for 50 min
[0105] FIG. 8:
[0106] Gel analysis of MS2-RNA which was isolated after incubation
in serum/stabilization solution for 3 days at 40.degree. C. The GTC
content of the stabilization solution after serum addition in which
the relevant RNA sample was incubated is indicated in the
corresponding column. Column 1: 2.70 M GTC, column 2: 2.5 M GTC,
column 3: 2.36 M GTC, column 4: 2.20 M GTC, column 5: 2.08 M GTC,
column 6: 1.94 M GTC, column 7: 1.80 M GTC, column 8: 1.66 M
GTC.
[0107] FIG. 9:
[0108] Gel analysis of the PCR amplificates of MS2-RNA which was
isolated after 1 day and 8 days, respectively, of incubation at
40.degree. C. in serum/stabilization solution.
[0109] Column 1: Amplificate of the RNA isolated after 1 day,
column 2: amplificate of the RNA isolated after 8 days, column 3:
DNA marker, column 4: MS2-RNA positive control: 0.8 .mu.g in 10
.mu.l RT, 1:50 diluted, 1 .mu.l amplified.
[0110] FIG. 10:
[0111] Gel analysis of isolated MS2-RNA after 6 days (column 2-12)
and 13 days (column 14-19), respectively, of incubation at room
temperature in serum/stabilization solution. The pH which was
obtained after mixing of serum and stabilization solution is
written behind the corresponding columns.
[0112] Column 1, 13, 20: DNA marker, column 2: pH 8.0, column 3: pH
7.7, column 4: pH 7.5, column 5: pH 7.35, column 6: pH 7.18, column
7,14: pH 7.07, column 8,15: pH 6.94, column 9,16: pH 6.8, column
10,17: pH 6.72, column 11,18: pH 6.68 and column 12,19: pH 6.7. The
stabilization solution of RNA in column 12,19 had the same pH as
that of the RNA in column 11, but contained 5 m GTC instead of 4
M.
* * * * *