HK1261251A1 - Device and method for isolating nucleic acids from whole blood - Google Patents

Description

Device and method for separating nucleic acids from whole blood

The present invention relates to a method for the stabilization of nucleic acids, in particular RNA (including RNA contained in cells), from a biological sample, in particular from whole blood, preferably with subsequent isolation of the nucleic acids, as well as to a device for use as a blood collection tube comprising a composition suitable for the stabilization of total nucleic acids, in particular total RNA, and for subsequent isolation.

Compositions, methods for stabilizing nucleic acids from biological samples, in particular whole blood, and blood collection tubes comprising the compositions useful in the methods are characterized in that an efficient stabilization of nucleic acids, in particular RNA, in whole blood is achieved and allows for a fast and efficient separation method (process). Herein, stabilization includes all nucleic acids, in particular all RNA (including nucleic acids, in particular RNA, contained in cell-free plasma, and nucleic acids, in particular RNA, contained in cells present in blood).

Prior Art

WO02/056030 a2 describes a container for stabilizing the RNA content in a sample with a predetermined vacuum to aspirate a predetermined sample volume, wherein in the container an agent for inhibiting gene induction and counteracting enzymatic degradation of nucleic acids (Mittel) is provided, in particular mercaptoethanol, Dithiothreitol (DTT) and chaotropic salts (chaotropic salts), in particular guanidinium isothiocyanate or guanidinium hydrochloride.

WO 00/09746a1 describes a blood collection container comprising a solution for lysing (Lyse) cells and stabilizing nucleic acids with guanidinium salts, buffer, detergent and reducing agents, in particular DTT, β -mercaptoethanol and TCEP (tris (2-carboxyethyl) phosphine). Wherein a reducing agent, in particular beta-mercaptoethanol or DTT, is considered necessary for stabilizing RNA in serum.

WO2009/018034 a1 relates to a method for increasing the solubility of SDS in a lysis buffer for cells by adding a non-ionic detergent and for isolating DNA from animal tissue by adding a lysis buffer consisting of: 2M NaCl, 1.2% SDS, 12mM EDTA, 24mM Tris-HCl (Tris-HCl), pH 8.0, with 2% Tween (Tween polyoxyethylene sorbitan fatty acid ester), the extraction buffer consisting of: 50mM Tris-HCl, pH 7, 10mM EDTA, 7M guanidine hydrochloride, 5% Tween 20.

WO2007/060248a1 relates to lysis of cells, i.e. liver tissue, with chaotropic salts and subsequent addition of non-chaotropic salts to bind nucleic acids to a carrier material.

DE 10147439A 1 describes the addition of a lysis reagent for the purpose of separating DNA from blood, and then the separation of the DNA-containing cellular components by centrifugation. The precipitated DNA is purified by resuspension with guanidine hydrochloride and removal of impurities such as proteins and precipitated and isolated by addition of alcohol.

DE 102014220090B 3 describes a container for collecting a biological sample containing nucleic acids, into which a pharyngeal swab (abscichthysupfer) can be introduced, wherein the container contains a lysis solution.

CN 104673623 a describes a composition for stabilizing blood cell nucleic acids and cell-free DNA according to the english abstract for sample containers with lids.

Object of the Invention

It is an object of the present invention to provide alternative compositions and preferably alternative methods for stabilizing all nucleic acids in whole blood and then separating from whole blood. Preferably, the composition should stabilize the RNA in whole blood and allow a rapid method for isolating all nucleic acids, in particular RNA. More preferably, the composition should allow for nucleic acid stabilization of variable blood volumes (blood volume ) such that a blood collection tube containing the composition need not be configured to draw in a predetermined blood volume.

Disclosure of Invention

The invention utilizes the features of the claims, in particular by a composition for stabilizing all nucleic acids, in particular RNA, in whole blood and a method for stabilizing all nucleic acids in whole blood for subsequent isolation, which method is used simultaneously for the disintegration (or called disintegration or disintegration) of cells, so that when brought into contact with the composition, in particular during blood collection, the cellular nucleic acids are released and can be stably preserved with respect to nature and concentration and then isolated. The composition is characterized in that it prevents, in particular, the decomposition and the resynthesis of the RNA in the whole blood into which it is mixed. Accordingly, the present invention relates to the use of said composition, preferably the composition comprised in a blood collection tube, for said method, in particular as a means (reagent) for stabilizing and isolating all RNA in blood, without the need for a step of precipitation and isolation of RNA prior to adsorption of the RNA to an adsorbent. For separation, a mixture of blood and an aqueous composition may be contacted with an adsorbent without precipitating RNA. The isolation of RNA by contacting the mixture of aqueous composition and blood with the adsorbent can be performed directly from the complete (thorough) mixing of the aqueous composition and the blood, i.e. e.g. without precipitation and separation of nucleic acids from the mixture, optionally after storage during which the mixture is transported e.g. from the blood collection site to the site of the analytical process.

The biological sample is preferably a liquid and optionally comprises human or animal cells. The biological sample may be, for example, a body fluid, such as cerebrospinal fluid, urine, sputum, a biopsy containing individual or homogeneous cells and in particular whole blood (also referred to as blood). The present specification relates to whole blood as a representative of a biological sample.

The composition has or consists of, in the form of an aqueous solution:

at least one guanidine salt, preferably guanidine thiocyanate;

at least one buffer substance, preferably 2- (N-morpholino) ethanesulfonic acid (MES);

a non-ionic detergent, preferably Triton X-100; and

a complexing agent for divalent cations, preferably ethylenediaminetetraacetic acid (EDTA);

and in particular free of reducing agents, e.g. free of thiol compounds or precursor compounds thereof, in particular free of beta-mercaptoethanol and Dithiothreitol (DTT).

In the form of an aqueous solution, the composition has or consists of:

at least one guanidinium salt, for example up to 1.8 to 2.6M, preferably 2.0 to 2.4M, more preferably 2.2M;

at least one buffer substance to buffer the blood volume to a pH value of 5.0 to 8.0, such as a concentration of pH 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0 to 7.5, 7.6, 7.7, 7.8, 7.9, preferably pH 6.5 to 7.3, such as MES of at least 50mM, preferably at least 70mM, such as up to 100mM or up to 74 mM;

a non-ionic detergent, such as Triton X-100, up to 10 to 20% (w/v), preferably 12 to 18% (w/v);

for example 500 to 100mM, preferably 70 to 80mM, more preferably about 72mM, of a complexing agent for divalent cations, in particular EDTA,

for example, to achieve the following ratio of blood volume to volume of the aqueous composition: 1:13 or 1:3 or up to 1:2, preferably up to 1:2.6 or up to 1:2.5 or 1: 2.4.

Preferably, the present invention relates to the use of said composition: as a lytic agent for cells contained in a biological sample, in particular whole blood, as a stabilizing agent for all nucleic acids of the biological sample and for separation by adsorption of nucleic acids from a mixture of the biological sample and the composition onto a nucleic acid adsorbent, wherein the composition comprises or consists of, in the form of an aqueous solution:

a. at least one guanidine salt, wherein the guanidine salt is selected from the group consisting of,

b. at least one buffer substance for buffering to a pH of 5.0-8.0,

c. at least one nonionic detergent and

d. at least one complexing agent for divalent cations and is

e. Does not contain a reducing agent.

Preferably, the composition consists of an aqueous solution of:

a. at least one guanidine salt, wherein the guanidine salt is selected from the group consisting of,

b. at least one buffer substance, at least one of which,

c. at least one nonionic detergent and

d. at least one complexing agent

And optionally a protease and/or a DNase.

The composition can be used as a lytic agent for cells contained in whole blood, and can be used as a stabilizer for all nucleic acids contained therein and can be used for separation by adsorbing the nucleic acids directly from a mixture of a biological sample and the composition onto an adsorbent for the nucleic acids.

The buffer substance is for example selected from: tris (hydroxymethyl) aminomethane), citrate or dihydrate phosphate (disodium hydrogen phosphate) or dihydrate phosphate (sodium hydrogen phosphate), BisTris-buffer (bis (2-hydroxymethyl) -imino-tris- (hydroxymethyl) -methane), ACES (N- (2-acetamido) -2-aminomethanesulfonic acid), sodium bicarbonate, preferably 2- (N-morpholino) ethanesulfonic acid (MES).

The nonionic detergent is for example selected from: tween 20 (polyoxyethylene (20) sorbitan monolaurate), Nonidet P40 (4-nonylphenol-polyethylene glycol), tween 80 (polyoxyethylene sorbitan monooleate), Brij 58 (polyethylene glycol cetyl ether), Triton X-114 (octylphenol polyglycol ether), preferably Triton X-100 (polyethylene glycol-P- (1,1,3, 3-tetramethylbutyl) phenyl ether).

The composition has the following advantages: the human cells contained in the whole blood are lysed and all nucleic acids, in particular RNA, from the whole blood is stabilized, wherein the whole blood and the composition may be present in variable volume ratios. Thus, a blood collection tube comprising the composition may be configured to aspirate variable volumes of blood, e.g. the blood collection tube may be configured to aspirate a volume ratio of blood to the composition of up to 1:13 or 1:3, preferably up to 1:2 or up to 1: 2.6. To aspirate variable volumes of blood, the blood collection tube containing the composition may have, for example, a piston that can be manually removed from the tube.

The composition is characterized in that it does not require precipitation of nucleic acids from a mixture of whole blood and the composition to stabilize and isolate nucleic acids, in particular RNA, in whole blood. Thus, the method according to the invention preferably comprises isolating nucleic acids, in particular RNA, from a mixture of whole blood and the composition without a step of precipitating the nucleic acids, in particular RNA, or without a centrifugation step for isolating the precipitated nucleic acids prior to binding the nucleic acids, in particular RNA, to the adsorbent.

According to the invention, preferably all RNA is isolated from a mixture of whole blood and the composition as follows:

optionally after addition of a protease, e.g.proteinase K, and incubation for e.g.at room temperature for about 15 minutes, optionally before or after addition of the protease, adding a DNase (DNase), contacting the mixture of whole blood and composition with an adsorbent for nucleic acids,

wherein no components are separated from the mixture (optionally including added protease and/or DNase) prior to contact with the adsorbent,

the material not bound to the adsorbent is then removed, for example by washing the adsorbent,

and eluting the nucleic acid bound to the adsorbent, for example by contacting the adsorbent with an aqueous buffer having a pH, an alcohol content, and/or an ion concentration that solubilizes the nucleic acid bound to the adsorbent.

The mixture of whole blood and the composition may be contacted with the adsorbent for nucleic acid completely and directly after preparing the mixture in terms of time. This leads to the advantage that the method for isolating nucleic acids, in particular RNA, does not require incubation for precipitation and does not require centrifugation before contacting the mixture with the adsorbent, and does not require the addition of further substances and can therefore be carried out easily and quickly.

After contacting the mixture of whole blood and composition (optionally including added protease), dnase may optionally be added to digest the DNA and substantially isolate RNA.

Adsorbents for nucleic acids are, for example, silica surfaces, in particular silica (gel) membranes, silica (gel) suspensions or silica-coated magnetic particles, which can be obtained, for example, under the following names for the isolation of nucleic acids: PAXgene of PreAnalytiX, NucleoSpin of Macherey-Nagel, mRNA Isolation Kit for Blood/Bone Marrow of Roche (mRNA Isolation Kit for Blood/Bone Marrow), or High purity Viral Nucleic Acid High Volume Kit (High Pure Viral Nucleic Acid Large Volume Kit).

When the adsorbent consists of silica-coated particles, preferably magnetic particles, an effective volume of diluent is preferably added to the mixture of whole blood and composition. The diluent may be, for example, a DNA/RNA stabilizing reagent for blood/bone marrow obtained from Roche corporation (Roche product No. 11934317001) or a composition according to the invention, in each case optionally having an ethanol content of up to 30 vol/vol%, preferably an ethanol content of 10 to 30 vol/vol%, for example an ethanol content of 20 vol/vol%. The effective volume may be determined such that substantially at least 80% of the total amount of RNA is adsorbed to the silica-coated particle, preferably substantially completely adsorbed to the silica-coated particle. The effective volume of diluent added to the mixture of whole blood and composition is preferably 50 to 150%, for example 80 to 120%, preferably 100% of the volume of the mixture added before or after mixing the silica-coated particles, which are preferably magnetic particles, into the mixture. In particular, in this embodiment, it is preferred to add a protease to the mixture.

The Silica-Coated particles can be, for example, those of Chemagen or Applied Biosystems (RNA-binding beads, product No. 100191) or mRNA isolation kits for blood/bone marrow from Roche (product No. 11934333001) or those according to Hai N.H., Phu N.D., Luong N.H., Chau N.S., Chinh H.D., Honang L.H., Leslie-Pelecky D.L (2008), Mechanism for Magnetic Nanoparticles units amide compositions, Journal of the Korea Physical therapy, 52(5),1327-1331, or those according to Quy D.V, Hieu N.M., Trac P.T., Nam N.H., Hai N.H., Sonn.H., synthetic N.M., P.T. Trac P.T., Nanometric P.S.H., Hai N.H., S. N.S. S. No. 7, C. Pat. No. 3, C.S. P.S. 7, C.S. P.S. D.S. 7, C.S. Pat. 7, Journal of Magnetic Nanoparticles, N.S. Pat. 3, N.S. No. 7, N.S. 4, N.S. 7, N.S. A.

Even for an adsorbent consisting of silica-coated particles, which are preferably silica-coated magnetic particles, complete consisting of whole blood and a composition can be obtainedThe mixture is contacted with the adsorbent with the diluent added, i.e., directly or completely or without prior separation of the mixture components.

According to the present invention, the complete mixture may be contacted directly with the adsorbent, since no components or fractions are separated from the mixture of whole blood and composition, in particular, centrifugation of such mixture is not required to separate precipitated components, e.g., centrifugation to separate precipitated nucleic acids and subsequent separation of the liquid phase and solubilization of the nucleic acids is not required. This is presently due to the fact that the composition does not precipitate nucleic acids, in particular RNA, in a mixture with whole blood. In contrast, the composition has the following advantages: the mixture of whole blood and composition, optionally with added protease and/or dnase, may be contacted with an adsorbent for nucleic acids, in particular a silica surface, without further processing steps or without additives to bind the nucleic acids to the adsorbent. Thus, the composition allows for a method for isolating nucleic acids that proceeds without the need to separate the nucleic acid-containing fraction (e.g., without centrifugation for precipitation and subsequent solubilization) prior to contact with the adsorbent.

The composition stabilizes nucleic acids, in particular RNA, in a mixture of whole blood and the composition, for example upon storage for at least 3 days, preferably at least 5 days, such as up to 4 days, in particular without freezing, for example upon storage at least 0 ℃, such as at room temperature to 22.5 ℃. Thus, the method preferably comprises isolating nucleic acids, in particular RNA, from the mixture, e.g. after storage without freezing or after storage at least 0 ℃, e.g. at 15 to 25 ℃, in particular up to 22.5 ℃. Alternatively or additionally, the mixture may be stored frozen, for example at-40 ℃ or below.

The invention will now be described in more detail, by way of example, with reference to the accompanying drawings, which show:

FIG. 1 integrity of RNA isolated from whole blood mixed with the composition of the invention in various volume ratios,

FIG. 2 concentrations of RNA isolated from whole blood mixed with the composition of the invention in various volume ratios,

FIGS. 3 and 4 compare the RNA integrity of the whole blood samples after storage in a stabilizer according to WO 00/09746 with the whole blood samples in the case of storage in a composition according to the invention (Sp.1 (donor No. 1) and Sp.2 (donor No. 2)),

FIG. 5 Cluster analysis of the concentration of various mRNAs in total RNAs isolated from whole blood mixed with the composition of the present invention,

FIG. 6 is a correlation matrix representing the differences in expression of memory dependencies,

figures 7 and 8 show the relative amount of mRNA in two donor samples, isolated from whole blood mixed with the composition of the invention after 5 days of storage, determined by RT-PCR.

The composition of the invention is in the form of an aqueous solution in a blood collection tube into which a blood sample is drawn. The blood collection tube can be directly used for collecting blood. Alternatively, the composition may be mixed with whole blood (which is preferably from a whole blood sample taken directly before). The disintegration (or so-called disintegration, disintegration) of the cells and the stabilization of nucleic acids, in particular RNA, of a blood sample is generally carried out by mixing the whole blood sample with a composition.

Example 1: isolation of RNA from Whole blood

As the composition of the present invention, an aqueous solution consisting of: 2.2M guanidinium thiocyanate, 72mM MES, 14.4% (w/v) Triton X-100, and 72mM EDTA in water. 6.5mL of the composition was contained in a conventional blood collection tube into which the freshly collected blood was drawn without further treatment and without additives. The whole blood was drawn into a blood collection tube in volumes of 0.5mL, 1.0mL, 1.5mL, 2.0mL, and 2.5mL, respectively. Mixing is carried out by aspiration of the blood volume, optionally additionally by shaking, in particular after the (Einziehen) bubbles have been aspirated.

Proteinase K (250. mu.L, 18. mu.g/mL) was added to the mixture of the composition and whole blood. The mixture was incubated at room temperature for about 15 minutes. Subsequently, the mixture was passed completely through a column (High Pure Viral Nucleic Acid Large Volume Kit, available from Roche) with an adsorbent for Nucleic acids. The adsorbent was washed with wash buffer according to the manufacturer's instructions and then the bound nucleic acids were eluted with 100. mu.L of elution buffer.

The quality of the eluted RNA was assessed by electrophoresis in an RNA nanochip system (Agilent Technologies, USA) and was detected using an Agilent 2100 Bioanalyzer, where the so-called RIN (RNA Integrity Number) (determined using the Bioanalyzer 2100 in the analysis) is given as a measure of RNA Integrity. FIG. 1 shows the results of the analysis of different volume ratios of whole blood to composition (RNA Exact): RNA integrity is independent of volume ratio, or for different volume ratios than whole blood, the composition stabilizes RNA of the same quality and RNA can be isolated from the mixture.

Fig. 2 shows the measured RNA concentrations in the eluates separated for different volume ratios. The results show that by increasing the RNA concentration in 100 μ Ι _ of eluate in proportion to the blood volume, the composition RNA (exact) stabilizes the RNA to the same extent for different volume ratios and separates it therefrom to the same extent. This indicates that the composition is suitable for lysing cells in whole blood and stabilizing nucleic acids in whole blood in various volume ratios.

Example 2: comparison of the stabilization Capacity of the compositions according to the invention with the comparative solutions

As composition according to the invention (RNA Exact), 6.5ml of the composition of example 1 were used in each case and mixed with 2.5ml of the previously directly drawn blood without further additives to prepare a mixture of composition and whole blood. For comparison, the following solutions were used (control, corresponding to WO 00/09746a 1): it consisted of 4.0M guanidinium thiocyanate, 45mM Tris/HCl, 18.0% (w/v) Triton X-100, and 0.8% (w/v) DTT in water and was adjusted to a pH of 6.0. 2.5ml of the comparison solution (as described in WO 00/09746A1 (ratio 1+1)) were mixed with the same 2.5ml of a previously directly drawn blood sample. The mixture was stored at 22.5 ℃ and RNA was isolated from the mixture using the NucleoSpin RNA Blood Midi Kit from Macherey-Nagel. To the comparative solution, 1/3 volumes of 70% ethanol were added as provided in the Macherey-Nagel's use Specification. This is not necessary for mixtures with the composition according to the invention. FIGS. 3 and 4 show the results of capillary electrophoresis separation of eluents on RNA nano-chips in Bioanalyzer 2100. It is possible to isolate RNA with high integrity from the sample mixture with the composition according to the invention even after 3 days and after 5 days, which is only possible with the use of the comparative solution at day 0, i.e. directly after mixing with blood.

Example 3: stabilization and isolation of RNA in whole blood

As a composition of the invention, 6.5mL each of the composition of example 1 was used and mixed with 2.5mL of the blood directly collected before (which contained no additional additives) to prepare a mixture of the composition and whole blood. For comparison, blood was drawn into a blood collection tube containing EDTA for inhibition of coagulation (S-monovites EDTA-K3, available from Sarstedt corporation). The blood was from three different donors, respectively.

The mixture according to the invention and the comparative sample were incubated at 22.5 ℃, with aliquots being taken immediately (T0, day 0), after 1 day of incubation (T1) and 3 days of incubation (T3). The nucleic acids were isolated directly and immediately from the removed aliquot as described in example 1.

The eluted nucleic acid was treated with DNase (Thermo Fisher Scientific, product number EN0521) to remove DNA, and the concentration of about 47,000 transcripts (mRNA) was determined by microarray analysis (humanHT-12 v4.0expression Bead Chip of Agilent). In the assessment, the difference in the number of transcripts was first shown to be more than 2-fold for the same total amount of RNA. Thus, in fig. 5, equal intensities in the matrix show a significant number of transcripts. Fig. 5 shows transcript numbers for three sample sets: stabil (stable) (composition according to the invention), unbeh (mixed with EDTA only) and T0 (no additives, isolated immediately after blood collection), directed against samples of donors 1,2 and 3, respectively, at the indicated time points.

The aliquots analyzed in fig. 5 were:

where EDTA-comparative sample is labeled "K".

Cluster analysis of 199 exemplary selected transcripts (shown in fig. 5) clearly showed that each of the blood samples mixed with the composition according to the invention (Stabil) for the donors at different time points of incubation showed comparable transcript patterns, whereas the control sample with EDTA alone (unbeh) showed a large difference within each donor at different time points of incubation, indicating the change in transcript numbers with time and with respect to T0.

For EDTA-only blood samples, concentration differences were found for 768 transcripts on day 3 relative to day 0, whereas samples incorporating the inventive compositions, within each donor, individual RNAs of the samples analyzed at later time points were found to differ in concentration little to no difference compared to T0 (see correlation matrix of fig. 6, comparative sample K, KT0, KT1, KT3 at each time point, with the inventive compositions T0, T1, T3).

The results show that the composition of the invention stabilizes the individual RNA molecules from whole blood during incubation, in terms of a constant number of transcripts of a single donor over a period of 3 days in an aliquot of a blood sample mixed with the composition, and that the composition significantly reduces denaturation and/or induction of mRNA, for example, compared to the EDTA control sample.

Example 4: reverse transcription and PCR based on RNA from whole blood

Up to 2.5mL of whole blood from two different donors each, was mixed directly after collection with 6.5mL of a composition of the invention consisting of 2.2M guanidinium thiocyanate, 72mM MES, 14.4% (w/v) Triton X-100 and 72mM EDTA in water and incubated at 22.5 ℃. Aliquots were taken from these mixtures immediately (T0) and after 3 days (T3), after 4 days (T4) and after 5 days (T5) and frozen at-80 ℃. Subsequently, all samples were thawed in parallel and placed on a nucleic acid adsorbent (NucleoSpin RNA Blood, Macherey-Nagel). The adsorbent was washed and the nucleic acids eluted according to the manufacturer's instructions. DNase (DNase I, No RNAse, product No. EN0521, Thermo Fisher Scientific) was added to the eluate. RNA was reverse transcribed using "first strand cDNA synthesis kit" (product No. K1612, Thermo Fisher Scientific) to obtain cDNA. The product was amplified and quantified by real-time PCR using Maxima SYBR Green Green/ROX qPCR Master Mix (product No. Nr. # K0222, Thermo Fisher Scientific) as a replicate (Doppler ansatz) with primers specific for the nucleotide sequences of the β -actin, GAPDH, IL-8, c-Fos, IL-1B and TNF- α genes.

For evaluation, Ct values obtained for cDNA for β -actin and GAPDH were used as internal standards to which the Ct values measured for IL-8, c-Fos, IL-1B and TNF- α were then normalized. In addition, these relative concentrations of the cDNAs for IL-8, c-Fos, IL-1B and TNF- α at T3, T4 and T5 were correlated with their respective concentrations at T0. These results are called ddCts, and FIG. 7 shows the blood of one donor and FIG. 8 shows the blood of another donor. The results for both samples show that the composition of the invention stably maintained the relative concentration of cellular mRNA specifically detected in whole blood over an incubation period of 22.5 ℃.

Claims (17)

1. Use of a composition as a lytic agent for cells contained in whole blood, as a stabilizing agent for all RNA of whole blood and for separation by adsorption of RNA from a mixture of whole blood and said composition onto an adsorbent for nucleic acids, wherein said composition is in the form of an aqueous solution consisting of:

a. at least one guanidine salt, wherein the guanidine salt is selected from the group consisting of,

b. at least one buffer substance for buffering to a pH of 5.0-8.0,

c. at least one nonionic detergent and

d. complexing agent for at least one divalent cation

e. And optionally protease and/or DNase, and is

f. The method does not contain a reducing agent, and does not contain the reducing agent,

characterized in that the use is as a stabilizer for the total RNA contained in whole blood and for the separation by adsorption of nucleic acids directly from a mixture of a biological sample and the composition onto an adsorbent for nucleic acids, wherein the composition is provided in the mixture in a variable volume ratio of biological sample to the composition of up to 1:13, and the adsorption is carried out directly from the mixture as follows: without separating any components from the mixture, optionally adding a protease and/or dnase to the mixture, and without precipitating and separating nucleic acids from the mixture prior to contacting the nucleic acids with the adsorbent.

2. Use according to claim 1, characterized in that the guanidinium salt is guanidinium thiocyanate, the buffer substance is 2- (N-morpholino) ethanesulfonic acid (MES), tris (hydroxymethyl) aminomethane), citrate, phosphate (dibasic sodium phosphate) dihydrate, BisTris-buffer (bis (2-hydroxymethyl) -imino-tris- (hydroxymethyl) -methane), ACES (N- (2-acetamido) -2-aminomethane sulfonic acid), sodium bicarbonate or phosphate (dibasic sodium phosphate) dihydrate, the non-ionic detergent is Triton X-100, Tween 20, Tween 80 (polyoxyethylene sorbitan monooleate), Brij 58 (polyethylene glycol cetyl ether), Triton X-114 (octylphenol polyglycol ether) or Nonidet P40, and the complexing agent is ethylenediaminetetraacetic acid (EDTA), and it does not contain a thiol compound or a precursor compound thereof as a reducing agent.

3. Use according to claim 1 or 2, characterized in that it comprises separating nucleic acids from a mixture consisting of whole blood and the composition by contacting the mixture with an adsorbent for nucleic acids without previously precipitating and separating nucleic acids by centrifuging the mixture.

4. Use according to claim 1 or 2, wherein the composition is contained in a blood collection tube.

5. Use according to claim 3, wherein the composition is contained in a blood collection tube.

6. The use of claim 4, wherein the blood collection tube is configured to aspirate a variable volume of biological sample.

7. The use of claim 5, wherein the blood collection tube is configured to aspirate a variable volume of a biological sample.

8. Use of a blood collection tube containing a composition for use as a lytic agent for cells contained in whole blood, as a stabilizing agent for all RNA of whole blood and for separation by adsorption of RNA from a mixture of whole blood and the composition onto an adsorbent for nucleic acids, directly for blood collection, characterized in that the blood collection tube is configured to aspirate a variable volume of a biological sample and the composition is in the form of an aqueous solution consisting of:

a. at least one guanidine salt, wherein the guanidine salt is selected from the group consisting of,

b. at least one buffer substance for buffering to a pH of 5.0-8.0,

c. at least one nonionic detergent and

d. at least one complexing agent for divalent cations

e. And optionally protease and/or DNase, and is

f. The catalyst does not contain a reducing agent, and has the advantages of no reducing agent,

characterized in that the use of the lytic agent is as a stabilizer for all RNA contained in whole blood and for separation by adsorption of nucleic acids directly from a mixture of a biological sample and the composition onto an adsorbent for nucleic acids, wherein the composition is provided in the mixture at a variable volume ratio of biological sample to composition of up to 1:13 and the adsorption is performed directly from the mixture as follows: without separating any components from the mixture, optionally adding a protease and/or dnase to the mixture, and without precipitating and separating nucleic acids from the mixture prior to contacting the nucleic acids with the adsorbent.

9. Method for isolating total RNA in whole blood, characterized in that whole blood is mixed with a composition consisting of, in the form of an aqueous solution:

a. at least one guanidine salt, wherein the guanidine salt is selected from the group consisting of,

b. at least one buffer substance

c. At least one nonionic detergent and

d. at least one kind of complexing agent is added,

e. optionally a protease and/or DNase

Consisting of and being free of reducing agents that prevent degradation and re-synthesis of RNA in whole blood;

preparing a mixture consisting of whole blood and the composition in a volume ratio of blood to composition of up to 1:13, optionally storing the resulting mixture above 0 ℃, optionally adding protease and/or dnase to the mixture;

contacting the mixture with an adsorbent for nucleic acids, wherein no component is separated from the mixture prior to contact with the adsorbent;

removing unbound material from the adsorbent;

eluting RNA from the adsorbent.

10. The method of claim 9, wherein contacting the mixture with the adsorbent is performed without prior precipitation, separation, and solubilization of nucleic acids from the mixture.

11. The method according to claim 9 or 10, wherein the storage of the mixture is carried out at a temperature of at most 25 ℃ for at least 3 days.

12. The method according to claim 9 or 10, characterized in that the mixing of the biological sample with the composition is carried out in a volume ratio of up to 1: 13.

13. The method according to claim 12, characterized in that the mixing of the biological sample with the composition is carried out in a volume ratio of up to 1:2.

14. The method according to claim 11, characterized in that the mixing of the biological sample with the composition is carried out in a volume ratio of up to 1: 13.

15. The method according to claim 14, characterized in that the mixing of the biological sample with the composition is carried out in a volume ratio of up to 1:2.

16. The method of claim 9 or 10, wherein the adsorbent is a silica-coated particle and a diluent is added to the mixture of biological sample and the composition in a volume of at least 50% of the volume of the mixture.

17. The method of claim 11, wherein the adsorbent is a silica-coated particle and a diluent is added to the mixture of biological sample and the composition in a volume of at least 50% of the volume of the mixture.