EP2576820A1

EP2576820A1 - Stabilization of nucleic acids in cell material-containing biological samples

Info

Publication number: EP2576820A1
Application number: EP11722475.8A
Authority: EP
Inventors: Christoph Erbacher; Markus Kirchmann; Patrick Baumhof; Petrina Schick
Original assignee: Qiagen GmbH
Current assignee: Qiagen GmbH
Priority date: 2010-06-02
Filing date: 2011-06-01
Publication date: 2013-04-10
Also published as: WO2011151427A1; US20130137586A1

Abstract

The present invention relates to the use of an aqueous system for stabilization of cell material-containing biological samples with preservation of the cell morphology of the cell material, and to a process for stabilization of nucleic acids in cell material-containing biological samples with preservation of the cell morphology of the cell material.

Description

Stabilization of nucleic acids in cell-containing biological samples

The present invention relates to the use of a buffer for stabilizing cell material-containing biological samples to obtain the cell morphology of the cell material and a method for stabilizing nucleic acids in cell material-containing biological samples to obtain the cell morphology of the cell material.

The stabilization of nucleic acids in biological samples plays an increasingly important role in biological, medical and pharmacological research and diagnostics, as by studying the nucleic acids of a cell their genetic origin and functional activity can be determined. The investigation of the ribonucleic acids (RNA), in particular the so-called messenger RNA (mRNA) of a cell allows a direct determination of the gene activity of this cell by means of a gene expression analysis, which can provide a direct insight into the activity of the cell at the time of sampling, especially of mRNAs are present in the cell at the time of transcription of the genes transcribed at that time. A quantitative analysis of the mRNA of a cell by means of modern molecular biological methods such as the quantitative or real-time reverse transcriptase polymerase chain reaction (qRT-PCR or real-time RT-PCR) or gene expression chip analyzes, for example, allows the recognition of expressed genes for detection of infections, metabolic diseases or cancer. The analysis of the deoxyribonucleic acids (DNA) of a cell by means of molecular biological methods such as PCR or sequencing allows the determination of genetic markers and the detection of genetic defects. The analysis of genomic RNA and DNA can also be used for the direct detection of infectious agents such as viruses, bacteria, etc.

An indispensable prerequisite for such techniques of nucleic acid analysis is the immediate stabilization of the nucleic acids immediately after the removal of a biological sample from their natural environment. This applies in particular to RNA already immediately after the removal of the can be degraded from their environment by the ubiquitous occurring very stable ribonucleases (RNases). Since RNases are very active enzymes that, unlike DNases, do not require cofactors, even the smallest amounts of these enzymes are enough to break down a large part of the RNA contained in a sample within a very short time.

Furthermore, the expression pattern of a cell undergoes a rapid turn-over so that the cell can respond to a change in external circumstances. The expression pattern can change rapidly as soon as the sample is collected. By lowering the temperature, changing the gas equilibrium and diluting the sample by anticoagulants, which should prevent the coagulation of the sample, especially in blood samples, for example, by the induction of stress genes changes the expression pattern of the individual cells immediately after removal , Only if it is possible to prevent the ex vivo gene induction, in a sample taken from their natural environment, the in vivo transcription profile can be conserved and analyzed. Stabilization of the nucleic acids is therefore of immense importance, especially in the case of medical samples, which are often taken at one place, for example in a doctor's office and analyzed in a laboratory only after prolonged storage and transport.

Various approaches for the stabilization of nucleic acids are known from the prior art. The stabilization of RNA in tissue is achieved, for example, by ammonium sulfate, which is able to diffuse rapidly into the cells, where it reduces the transcription and degradation of RNA by RNases by denaturing cellular proteins (so-called RNAIater technology). However, this principle can not be applied to whole blood, which is one of the most important samples, because whole blood samples contain a high proportion of protein which forms an insoluble precipitate with the reagent. Another common method for stabilizing RNA from tissue samples is the use of guanidinium thiocyanate and β-mercaptoethanol, which lyses the cells and denatures the proteins contained therein (Gillespie, D., et al., Nucleic Acids Research, 1992, 20 (20), 5492). , Method for the stabilization of RNA in blood, which in addition to a large proportion of DNA and proteins in particular contains various intra- and extracellular RNases, currently based on the lysis of the cells and subsequent denaturation of RNases (US 06602718 B1, US 06617170 B1 and US 6821789). However, a significant disadvantage of these methods, especially in the case of blood, is that the lysis of the cells mixes the RNA of different cell types and thus a large background of undesired RNA subsequently makes it difficult to study the desired RNA. Specifically, in the case of blood, this is the high level of mRNA encoding hemoglobin derived from red blood cells that are about 1000 times more abundant than leukocytes.

The object of the present invention was therefore to provide a buffer for the stabilization of nucleic acids in cell-containing biological samples to obtain the cell morphology in order to enable a subsequent cell separation to stabilize the nucleic acids contained in the sample to the molecular " Actual state "at the time of sampling as possible, and also the proportion of unwanted

Nucleic acids, in particular unwanted RNA to reduce dramatically in the sample to be examined after the subsequent lysis of the cells.

Surprisingly, it has been found that an aqueous system comprising one or more substances selected from the group consisting of 3 - (/ V-morpholino) propanesulfonic acid (MOPS), 1, 2-dimethoxyethane, sodium salicylate, hexaammonium heptamolybdate, glucosamine hydrochloride, indole , 2- (4-hydroxyphenyl) ethanol and tetrahexylammonium chloride, nucleic acids stabilized in cell-containing biological samples to obtain the cell morphology of the cell material. According to the invention, "aqueous system" can be any water-based solution or buffer suitable for suspending or dissolving portions of cell material without denaturing components of the sample as long as the solution / the buffer contains at least one of the substances mentioned. proper aqueous system is able to stabilize both intracellular and extracellular nucleic acids in the presence of cell material. The solutions / buffers according to the invention are also suitable for the stabilization of

Nucleic acids in the presence of free (extracellular) nucleases (DNases and RNases).

The aqueous system preferably comprises MOPS or a mixture of MOPS and at least one further substance selected from the group comprising 1, 2-dimethoxyethane, sodium salicylate, hexaammonium heptamolybdate, glucosamine hydrochloride, indole, 2- (4-hydroxyphenyl) ethanol and tetrahexylammo includes chloride. Most preferably, the aqueous system comprises MOPS or a mixture of MOPS with sodium salicylate and / or glucosamine hydrochloride.

The concentration of the substance used for stabilization is dependent on the substance used. The concentration of 3 - (/ V-morpholino) propanesulfonic acid in the aqueous system according to the invention is preferably 1 to 1000 mmol / l, more preferably 25 to 500 mmol / l, further preferably 100 to 300 mmol / l and especially 200 mmol / l.

Contains the buffer sodium salicylate, the concentration of the

Sodium salicylate in the buffer preferably 1 - 1000 mg / ml, more preferably 25 to 500 mg / ml, further preferably 200 to 300 mg / ml and in particular 250 mg / ml. If the buffer contains hexaammonium heptamolybdate, the concentration of hexaammonium heptamolybdate in the buffer is preferably 1 to 300 mg / ml, more preferably 50 to 250 mg / ml, further preferably 100 to 200 mg / ml and especially 150 mg / ml. If the buffer contains glucosamine hydrochloride, the concentration of the glucosamine hydrochloride is preferably 1 to 300 mg / ml, more preferably 25 to 200 mg / ml, further preferably 50 to 150 mg / ml and especially 100 mg / ml. If the buffer contains indole, the concentration of the indole is preferably 1 to 300 mg / ml, more preferably 25 to 200 mg / ml, further preferably 50 to 150 mg / ml and especially 100 mg / ml. If the buffer contains 2- (4-hydroxyphenyl) ethanol, the concentration of the indole is preferably 1 to 300 mg / ml, more preferably 25 to 200 mg / ml, further preferably 50 to 150 mg / ml and especially 100 mg / ml. If the buffer contains tetrahexylammonium chloride, the concentration of indole is preferably 0.1 to 50 mg / ml, more preferably 0.5 to 25 mg / ml, further preferably 1 to 10 mg / ml and especially 5 mg / ml. If the buffer contains 1, 2-dimethoxyethane, the proportion of the 1, 2-dimethoxyethane in the buffer is preferably 1 to 20 vol% (volume percent), preferably 5 to 15 vol% and especially 10 vol%, corresponding to a concentration of 1, 2-dimethoxyethane of preferably 8.7 to 174 mg / ml, particularly preferably 43.5 to 130.5 mg / ml and in particular 87 mg / ml. The substances mentioned in this paragraph are preferably present in the aqueous system in a concentration range of 1 to 3000 mg / ml, more preferably 25 to 2000 mg / ml, more preferably 50 to 1500 mg / ml and especially 400 to 850 mg / ml on the total concentration, before.

The inventive solution / buffer according to the invention may moreover comprise one or more further substances, preferably selected from the group comprising pH regulators such as acids or bases, anticoagulants and organic solvents. For example, the buffer may contain weakly basic salts such as sodium acetate, preferably in a concentration of 10 to 200 mmol / l, more preferably 25 to 75 mmol / l. Suitable anticoagulants are known to the person skilled in the art and include, for example, chelating agents such as heparin, citric acid, ethylenediamine / V, / V, / V ', / V-tetraacetic acid (EDTA) and 1,2-bis (2-aminophenoxy) ethane. / V, / V, / \ / ', / \ / -tetra acetic acid (BAPTA) in the form of the acid, an alkali metal salt or ester, which are capable of complexing divalent metal ions such as calcium ions. The concentration of such chelating agents in the buffer according to the invention is preferably 2 to 100 mmol / l, particularly preferably 2 to 50 mmol / l and in particular 5 to 15 mmol / l. The buffer of the invention may further contain organic solvents such as DMSO, preferably in a concentration of 10 to 250 mmol / l, more preferably from 50 to 200 mmol / l. The pH of the aqueous system according to the invention is preferably 3 to 7, more preferably 3.5 to 6.5, particularly preferably 4 to 6 and in particular 4.5 to 5.5.

For the purposes of the invention, any sample containing cells is referred to as the cell-material-containing biological sample. For example, the samples may be derived from animal or plant tissues, tissue or cell cultures, bone marrow, human and animal body fluids such as blood, serum, plasma, urine, semen, cerebrospinal fluid, sputum and smears, plants, plant parts and extracts, e.g. As juices, fungi, prokaryotic or eukaryotic microorganisms such as bacteria or yeasts, fossil or mummified samples, soil samples, sewage sludge, waste water and food are obtained. Preferably, the biological sample comprises blood, more preferably whole blood.

The term nucleic acids referred to in the context of the invention, both ribonucleic acids (RNA) and deoxyribonucleic acids (DNA). The abbreviations RNA and DNA in the sense of the invention designate both a single nucleic acid molecule (a nucleic acid) and a multiplicity of nucleic acids. Preferred nucleic acids according to the invention are all ribonucleic acids, in particular messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), heterogeneous nuclear RNA (hnRNA), so-called small nuclear RNA (snRNA), so-called. small-interfering RNA (siRNA), micro-RNA (miRNA) and so-called antisense RNA.

The aqueous system according to the invention is capable of stabilizing nucleic acids, in particular the unstable RNA in a sample for at least 24 hours at room temperature, preferably at room temperature for at least three days. The term room temperature for the purposes of the invention preferably comprises temperatures of 22 ± 3 ° C. The shelf life of the stabilized sample at temperatures below 20 ° C, for example at 2 to 8 ° C is correspondingly higher.

For the purposes of the invention, a sample is referred to as stabilized, if the integrity of the nucleic acids contained after storage at a given temperature for the indicated time is higher than the integrity of the nucleic acids in a (unstabilized) comparative sample, which originates from the same source and was removed and stored under identical conditions but without the addition of a stabilizing buffer. Preferably, the integrity of the nucleic acids in a sample, which is referred to as stabilized in the context of the invention, after storage for 3 days (t = 72 h) at room temperature at least 60% of the integrity of the nucleic acids at the time of addition with the buffer according to the invention (t = 0 h), more preferably at least 80% and especially at least 95%. Methods for determining the integrity of nucleic acids are known to those skilled in the art. In the case of RNA, the stated percentage values refer to the RNA Integrity Number (RIN), whose determination will be discussed in more detail later.

The buffers / solutions of the invention are further capable of minimizing ex vivo gene expression in the stabilized samples as compared to unstabilized samples, thus preserving the in vivo transcription profile. Thus, the state of the cells at the time of sampling can be largely retained and examined despite storage at a later date. In particular, since cells outside their natural environment can significantly alter the pattern of expression (and thus transscription), this stabilization of nucleic acids and the suppression of ex vivo gene expression offers the possibility of storing sampled samples.

Another object of the invention is a method for stabilizing nucleic acids in cell material-containing biological samples to obtain the cell morphology of the cell material to provide samples for at least one of the following methods for examining the nucleic acids contained in the sample, but not limited thereto be: PCR, RT-PCR, electrophoretic methods, microarray analysis, labeling, isolation and / or detection of the nucleic acids, comprising the displacement of the biological sample with a nucleic acid-stabilizing aqueous system according to the invention. The faster the sample is removed after removal from its natural environment with the aqueous system according to the invention, the lower the extent of degradation of the nucleic acids contained in the sample and the ex vivo gene induction or repression. Preferably, the sample is added immediately after removal from its natural environment with the buffer of the invention. In a preferred embodiment, the sample is transferred immediately after removal from its natural environment in a vessel containing the inventive aqueous system. The volume of the buffer / solution used depends on the sample. For the stabilization of whole blood samples, the sample is mixed with a volume of the buffer / solution which is preferably from 1.5 to 1 (by weight, more preferably from 2 to 5 times the volume of the whole blood sample.

The nucleic acids stabilized in the aqueous system according to the invention can be labeled, processed, isolated and detected following storage by known methods. For this purpose, the cell material contained in the sample is first lysed, and the nucleic acids released thereby are isolated by means of a suitable method and optionally purified. The methods suitable for this purpose are known to the person skilled in the art. Lysis of the cell material can be carried out, for example, in the commercially available QIAzol lysis reagent from Qiagen (Hilden, Germany) in accordance with QIAzol Handbook 10/2006. For example, by a phenol / chloroform extraction, the RNA contained in the lysed sample can be separated from the DNA and the proteins and precipitated by subsequent precipitation with isopropanol from the aqueous phase. The purification of the RNA can be carried out, for example, using the commercially available RNeasy kits from Qiagen, or else using any other suitable purification method.

The nucleic acids obtained can then be processed further, for example. In the case of RNA by reverse transcription RT-PCR method are transcribed and amplified, amplified in the case of DNA by PCR method and / or by electrophoretic methods such as Northern blotting (RNA) or Southern blotting (DNA) or so-called microarrays (Genchip analyzes) are examined. The term RT-PCR encompasses in the sense of the invention in particular in particular so-called quantitative RT-PCR methods (qRT-PCR or real-time RT-PCR), which allow a quantification of the obtained mRNA.

The integrity of the RNA obtained was determined by means of electrophoretic methods. On the one hand, classical gel electrophoreses were performed on denaturing agarose gels. In intact RNA, such a gel after staining with fluorescent dyes such as ethidium bromide or SYBR Green two intensely fluorescent, sharply separated bands corresponding to the ribosomal RNAs 28 S and 18 S, and possibly other bands of lower intensity. The ratio of the fluorescence intensity of the 28 S rRNA band to the 18 S rRNA band is approximately 2: 1 in the case of intact RNA.

Further, using the Agilent 2100 Bioanalyzer from Agilent, electrophoretic examinations of the RNA obtained were performed on microchips. An algorithm implemented in the Bioanalyzer software was used to determine the RNA Integrity Number (RIN), which is a system for quantifying RNA quality that takes into account a number of other factors in addition to the intensity of the 28 S and 18 S rRNA bands thus enabling a more reliable statement about the integrity of the RNA than is possible with a purely visual estimation of the intensity on a gel. On the basis of the ribosomal subunits 28 S to 18 S rRNA and their ratio including degraded breakdown products, the software determines a RIN value on a scale of 1 to 10. The numerical value 1 corresponds to a completely degraded RNA, while the numerical value 10 corresponds to a completely intact RNA. From the thus determined integrity of the rRNA is concluded that the integrity of the mRNA.

In addition, no qPCR inhibitors are introduced into the sample during the work-up or retained in it by the buffers according to the invention. Furthermore, the inventive buffers are capable of minimizing ex vivo gene induction in the stabilized samples. This was detected by means of qRT-PCR studies of the RNA, which was isolated after storage of the sample in the buffers according to the invention by known methods. to Quantification of the results was carried out using the AACT method known to the person skilled in the art, in which the expression of the target genes (in the present case c-fos and I L-1β) is normalized with that of an unregulated reference gene (in the present case, 18 S rRNA used).

The inventive method may further comprise a step for immunohistological labeling of individual cell types in a sample containing different cell types, prior to examination of the nucleic acids contained in the sample with the above-mentioned techniques such as PCR, RT-PCR, electrophoretic Methods or microarray analyzes is performed. Since the stabilizing buffers of the present invention preserve the cell morphology of the cell material, the present invention enables the immunohistological labeling, assay and / or separation of individual cell types before subsequent lysing of the cell material releases the nucleic acids contained in the cells. For example, even after storage at room temperature for several days, the cells can be labeled with specific antibodies and detected. Since in this way specific individual cell types can be separated from the multiplicity of further cell types contained in a biological sample, the subsequent examination of the nucleic acids of the desired cell type becomes significant, for example, by techniques such as PCR, RT-PCR, electrophoretic methods or microarray analyzes simplified.

Preferably, the immunohistological labeling is carried out with fluorescence-labeled antibodies that allow a UVA / is spectroscopic detection of the antigen-antibody conjugate.

In a preferred embodiment, the method further comprises a step for selecting and separating individual cell types from a sample containing different cell types, prior to the examination of the nucleic acids contained in the sample using the above-mentioned methods such as PCR, RT-PCR , electrophoretic methods or microarray analyzes. The method allows, for example, the flow cytometric analysis of the stabilized cell material. With the help of flow cytometry, a large number of cells can be characterized on a single cell level in terms of their biochemical and physical properties within a very short time. For this reason, this technique is routinely used inter alia in hematology and immunology, for example for the diagnosis and assessment of the course of the disease or the therapeutic success of various diseases and viral infections such as, for example, leukemia or HIV infections.

The principle of Durchflußzytometrie is based on the study of the optical properties of the cells that pass through a laser beam in a single measuring unit. On the one hand, photomultipliers are used to investigate the light scattering and refraction caused by a cell crossing the laser beam. The amount of scattered light depends on the size of the cell and its complexity. For example, granulocytes that have a rough surface scatter significantly more light than T lymphocytes that have a smooth surface. The forward scattered light (FSC) correlates with the volume of the cell, while the sidewards scatter (SSC), measured at an angle of 90 °, correlates with the granularity of the cell, the structure and size of its nucleus, and the amount of vesicles in the cell depends. Using these parameters, the cell types of the blood can be differentiated into granulocytes, lymphocytes and monocytes. Furthermore, the flow cytometry also allows the determination of the number of cells in a sample and a separation of the individual cell types.

In addition to the scattering in the flow cytometer but also the emission of fluorescent dyes can be detected and quantified. In fluorescence-based flow cytometry, often called fluorescence activated cell sorting (FACS), the cells are first labeled with a fluorescent dye that specifically binds to certain components of the cell prior to flow cytometric analysis. The intercalating dyes 4 ', 6-diamidino-2-phenylindole (DAPI) and For example, propidium bromide bind to the DNA of a cell and, by measuring the fluorescence intensity, make it possible to determine the DNA content of the cell. The cells can also be labeled with a specific antibody which either carries a fluorescent dye itself (direct immunofluorescence) or which is labeled in a second step after binding to the antigen with a fluorescently labeled secondary antibody (indirect

Immunofluorescence). For example, by using CD3 antibodies labeled with the fluorescent dye fluorescein isothiocyanate (FITC) (CD3-FITC antibodies), specifically mature T lymphocytes can be labeled and detected, counted and separated by flow cytometry. By using multiple laser sources, various features can also be detected simultaneously using multiple antibodies and used as selection criteria for subsequent separation of the labeled cells. In the method according to the invention, therefore, the selection and separation of the cell types preferably takes place by means of fluorescence-based flow cytometry.

Description of the pictures

FIG. 1 shows the electropherograms of two RNA samples which a) in an aqueous system according to the invention in the presence of lysed blood (left column) or b) in an unstabilized aqueous solution without lysed blood (positive control, right column). each incubated for 24 hours, 3 and 7 days at room temperature. The RNA integrity number (RIN), which was determined using a software-implemented algorithm, is also given in the respective electropherogram.

Figure 2 shows a comparison of the integrity of RNA isolated from blood samples stored for 2, 24 and 72 hours respectively in a pH 5 MOPS buffer and in commercially available PAXgenes or EDTA-containing sample vials has been. Figure 3 shows the relative c-fos transcriptional profile of qRT-PCR of a sample stored in an EDTA-containing buffer (upper panel) compared with that of a sample stored in a MOPS buffer of pH 5 was stored (lower diagram).

Figure 4 shows the relative IL-1β transcriptional profile of qRT-PCR of a sample stored in an EDTA-containing buffer (upper panel) compared to that of a sample stored in a MOPS buffer of the pH. Value 5 was stored (lower diagram).

Figure 5 shows the electrophoresis gels of RNA recovered from whole blood samples after storage at room temperature for 24 hours. On the far left is shown an image of the electrophoresis gel of RNA isolated from an unstabilized stored blood sample, while the RNA examined in gels II to V was stored in stabilizing buffers according to the invention (II: buffer containing MOPS, pH 5; III: buffer containing MOPS and 250 mg / ml sodium salicylate, pH 5; IV: buffer containing MOPS and 100 mg / ml glucosamine hydrochloride, pH5; V: buffer containing MOPS and

250 mg / ml sodium salicylate and 100 mg / ml glucosamine hydrochloride, pH 5).

FIG. 6 shows the scattered light dot plots obtained by flow cytometric investigations a) of a whole blood sample not stabilized according to the invention, which had been previously stored for 24 hours at 4 ° C. in a commercially available citrate buffer and b) a whole blood sample which was previously 24 h was stored in a pH 5 buffer according to the invention containing MOPS and 100 mg / ml glucosamine hydrochloride. On the left are the dot plots of the samples before staining with specific antibodies, in the middle the dot plots after labeling of the leukocytes with CD3-FITC, and on the right the histograms of the CD3-FITC-labeled leukocytes are shown. X-Axis: Forward Scatter (FSC) Y-Axis: Sideward Scatter (SSC) Examples

Example 1: Stabilization of RNA in the presence of lysed blood

The base buffer used was a solution of 200 mmol / l MOPS, 50 mmol / l sodium acetate and 10 mmol / l EDTA in RNase-free water (pH 5). This base buffer was admixed with the substances indicated in Table 1. 800 μΙ of the various buffer compositions were mixed with 6 g of RNA, which had previously been isolated from Jurkat cells using the commercially available RNeasy kit from Qiagen (Hilden, Germany), and after addition of lysed blood containing free activated RNases, incubated for a predetermined time. As a control (positive control), 6 g of RNA without lysed blood were incubated in an unstabilized aqueous solution for the same time period.

After each 1 hour, 1 day or 3, 5 and 7 days, the samples were lysed according to the QIAzol Handbook 10/2006 using the QIAzol lysis reagent from Qiagen (Hilden, Germany), and the RNA contained isolated using a RNeasy kit from Qiagen (Hilden, Germany). For this purpose, the sample was admixed with 2.5 ml of QIAzol reagent from Qiagen (Hilden, Germany) and 500 μl of chloroform and centrifuged for 15 minutes at 12,000 rpm. The upper phase was carefully removed and mixed with 2.5 ml of ethanol. To attach the RNA to the RNeasy mini-column from Qiagen (Hilden, Germany), the column was loaded with the lysate and centrifuged for 1 min at 8000 rpm. 500 μl RW1 buffer from Qiagen (Hilden, Germany) were pipetted onto the column and the column was centrifuged for 1 minute at 8000 rpm. 80 μM of a mixture of 10 μM Dnase I solution and 70 μM RDD buffer from Qiagen (Hilden, Germany) were pipetted onto the column and the column was incubated for 20 min at room temperature. Subsequently 500 μl of RW1 buffer from Qiagen (Hilden, Germany) were pipetted onto the column again and the column was centrifuged for 1 minute at 8000 rpm. The column was washed twice with 1 ml RPE buffer (1 min at 8,000 rpm) and then centrifuged for 10 min at 14,000 rpm to dry. To elute the RNA

5 100 μΙ Rnase-free water were pipetted onto the column, the column was incubated for 1 min at room temperature and then centrifuged for 1 min at 14000 rpm. The eluate contained the purified RNA. This was detected via a denaturing agarose / formaldehyde gel by staining with SYBR Green. Buffers suitable for stabilization were considered to be those in which, even after 3, preferably after 5, more preferably after 7 days of storage in the presence of lysed blood, both 28 S rRNA and 18 S rRNA were still detectable on the agarose gel. In particular, the buffers for which an intensity ratio of the two bands (28 S: 18 S) of about 2: 1 was retained were termed stabilizing. In particular, the solutions listed in Table 1 proved to be suitable for stabilizing RNA in the presence of free active RNases for longer than 24 h at room temperature.

Table 1

Stabilizing buffer composition

Buffer 1 base buffer + 10% by volume 1, 2-dimethoxyethane, pH 4

Buffer 2 250 mg sodium salicylate per ml of base buffer

Buffer 3 150 mg hexaammonium heptamolybdate per ml of base buffer

Buffer 4 100 mg glucosamine hydrochloride per ml of base buffer

Buffer 5 100 mg indole per ml of base buffer

Buffer 6 100 mg of 2- (4-hydroxyphenyl) ethanol per ml of base buffer

Buffer 7 5 mg tetrahexylammonium chloride per ml of base buffer Buffer 8 250 mg sodium salicylate and 100 mg glucosamine hydrochloride per ml of base buffer

The integrity of the RNA obtained was determined using the Bioanalyzer 2100 from Agilent Technologies (Böblingen, Germany). By way of example, a comparison of the sample stabilized in buffer 2 with the unstabilized sample (positive control) in FIG. 1 is shown. As early as 24 h, the electropherogram of the positive control shows a clear baseline noise between the 18 S and the 28 S rRNA bands at 43 s and 50 s, respectively (so-called interregion), which is characteristic of degradation of the RNA in the sample. The RIN of the positive control was only 9.0 after 24 h, while the RNA of the sample stabilized in buffer 2 according to the invention had a RIN of 10.0.

After three days storage of the samples at room temperature, the intensity of the 28 S band was already lower than the intensity of the 18 S band in the positive control, and the RIN was only 6.5. Only a small increase in noise was seen in the sample stabilized with the buffer of the present invention, and the RIN was still 10.0. Even after seven days of storage at room temperature, in the stabilized sample the 18 S and 28 S rRNA bands are still recognizable as separate bands with an intensity ratio of 28 S: 18 S = 1 .8 (RIN 6.8), while in the electropherogram the control is none discrete 28 S band was more noticeable (RIN 4.5). The values of the other buffer solutions listed in Table 1 gave a similarly good stabilization of the samples.

Example 2 Amplification of the Stabilized RNA by Quantitative RT-PCR [0047] To ensure that qPCR inhibitors are not introduced into the sample during the work-up by the stabilizing buffers according to the invention. or were retained in it or the substances used in the buffers themselves acted as inhibitors, and that the buffers of the invention are also capable of minimizing ex vivo gene induction, became a part of the RNA obtained using commercially available primers for GAPDH from the company Operon Biotechnologie Inc. (Huntsville, Alabama, USA) was amplified by a qRT-PCR according to a standard protocol of Applied Biosystems Inc. (Foster City, California, USA).

For this purpose, a portion of the RNA obtained in Example 1 (6 g) was mixed with 2.5 ml of lysed blood three different donors and then mixed with 5 ml of the base buffer or added to commercially available PAXgene sample containers and EDTA-containing sample containers. Immediately after mixing the samples with the buffer (0 h), and after storing the samples in the buffer for two hours (2 h), one or three days (24 h and 72 h, respectively) at room temperature, that in the samples contained RNA according to the QIAzol Handbook 10/2006 using the QIAzol lysis reagent and the RNeasy kit from Qiagen (Hilden, Germany) isolated. All investigations were performed as duplicate determinations.

The integrity of the resulting RNA from a donor (donor 3) was determined using the Agilent Bioanalyzer 2100 as described in Example 1. The results are shown in Figure 2. It was found that the integrity of the RNA obtained from the sample stored in the buffer according to the invention is higher than the integrity of the RNA isolated from the samples stored in PAXgene or in EDTA.

The amount of RNA isolated from 2.5 ml of blood was determined for the samples stored in the MOPS buffer or in EDTA after dilution with water (factor 7.5) by photometric determination of the light absorption at a wavelength of 260 nm. The purity of the RNA obtained is determined by photometric determination of the ratio of light absorption at 260 nm to that at 280 nm. The results are given in Table 2, with the mean values of the duplicate determinations given in each case. Table 2

Donor method time A260 / A280 total yield per 2.5 ml of blood

[h] [mg]

1 EDTA 0 2.1 4.36

2 2.05 8.55

24 2.2 9.44

72 2.1 5.97

MOPS pH 5 0 2.1 11.02

2 2.1 7.16

24 2.15 7.06

72 2.05 5.12

2 EDTA 0 1.9 5.45

2 1.95 4.26

24 2.05 3.40

72 1.9 2.94

MOPS pH 5 0 1.95 4.32

2 1.9 3.53

24 1.85 2.31 72 2.0 2.94

3 EDTA 0 2.0 12.05

2 2.05 10.16

24 2.1 7.99

72 2.0 6.17

MOPS pH 5 0 2.0 9.77

2 1 .85 3.47

24 2.1 1 1.22

72 2.0 6.63

The relative expression of c-fos and IL-1β was examined for the samples given in Table 2 by means of the AAC-r method relative to the expression of 18 S rRNA. The results are summarized in Tables 3 and 4 and illustrated in Figures 3 and 4 and Figures 5 and 6, respectively.

Table 3: Effect of storage conditions on the transcription of c-fos

Donor method time [h] C _T (c-fos) C _T (18 S AC _T (18S-ΔΔΟτ (to-tx) rRNA) c-fos)

1 EDTA 0 27.27 25.43 -1 .84 0

27.27 25.34 -1 .93 0

2 24.16 25.76 1 .60 -3.44 24.04 25.53 1.49 -3.42

24 22.47 25.83 3.36 -5.20

22.29 25.91 3.62 -5.55

72 27.89 27.48 -0.41 -1.43

25.71 25.86 0.15 -2.08

MOPS pH 5 0 26.89 25.88 -1.01 0

27.06 25.18 -1.88 0

2 26.63 25.37 -1.26 0.25

27.01 25.61 -1.40 -0.48

24 24.21 25.76 1.55 -2.56

24.70 25.46 0.76 -2.64

72 27.35 27.30 -0.05 -0.96

26.00 25.73 -0.27 -1.61

EDTA 0 26.76 25.43 -1.33 0

27.72 25.02 -2.70 0

2 24.78 25.18 0.40 -1.73

24.92 24.64 -0.28 -2.42

24 23.03 24.74 1.71 -3.04

23.12 24.17 1.05 -3.75

72 24.56 27.56 3.00 -4.33

24.68 24.75 0.07 -2.77

MOPS pH 5 0 27.17 24.66 -2.51 0

26.70 24.22 -2.48 0

2 27.19 24.94 -2.25 -0.26 27.76 24.52 -3.24 0.76

24 24.14 24.66 0.52 -3.03

24.18 24.79 0.61 -3.09

72 25.57 24.61 -0.96 -1 .55

25.51 25.30 -0.21 -2.27

EDTA 0 26.51 24.08 -2.43 0

27.45 25.02 -2.43 0

2 23.93 25.14 1 .21 -3.64

23:56 24.86 1 .30 -3.73

24 21.41 24.68 3.27 -5.70

21.97 25.03 3.06 -5.49

72 28.53 24.38 -4.15 1 .72

26.49 24.12 -2.37 -0.06

MOPS pH 5 0 27.20 25.52 -1 .68 0

27.51 25.27 -2.24 0

2 26.1 1 24.39 -1 .72 0.04

27.34 25.02 -2.32 0.08

24 23.98 24.77 0.79 -2.47

24.51 24.68 0.17 -2.41

72 25.69 25.14 -0.55 -1 .13

25.71 25.22 -0.49 -1 .75 Table 4: Effect of storage conditions on the transcription of IL-1β

Donor method time [h] C _T (IL-1β) C _T (18 S AC _T (18S-ΔΔΟτ (to-tx) rRNA) IL-1β)

1 EDTA 0 24.53 22.34 -2.19 0

24.68 22.30 -2.38 0

2 24.17 23.08 -1 .09 -1 .10

24.09 23.40 -0.69 -1 .69

24 25.1 1 22.49 -2.62 0.43

25.21 22.56 -2.65 0.27

72 29.13 23.31 -5.82 3.63

29.26 23.04 -6.22 3.84

MOPS pH 5 0 24.87 22.18 -2.69 0

25.08 22.51 -2.57 0

2 24.33 21.97 -2.36 -0.33

24.31 22.14 -2.17 -0.40

24 25.48 22.18 -3.30 0.61

25.35 22.89 -2.46 -0.1 1

72 27.34 24.13 -3.21 0.52

26.22 23.44 -2.78 0.21

2 EDTA 0 26.21 21.72 -4.49 0

26.29 22.08 -4.21 0

2 26.35 22.37 -3.98 -0.51

26.51 22.34 -4.17 -0.04 24 28.79 21.98 -6.81 2.32

28:43 22:19 -6.24 2.03

72 32.53 22.24 -10.29 5.60

32.28 23.84 -8.44 4.23

MOPS pH 5 0 25.51 21.54 -3.97 0

26.03 21.72 -4.31 0

2 25.80 21.81 -3.99 0.02

25.72 21.58 -4.14 -0.17

24 27.22 21.67 -5.55 1 .58

26.99 22.10 -4.89 0.58

72 27.53 21.57 -5.96 1 .99

28.02 21.55 -6.47 2.16

EDTA 0 24.84 23.64 -1 .20 0

25.45 23.43 -2.02 0

2 24.47 23.78 -0.69 -0.51

24.44 23.65 -0.79 -1 .23

24 26.43 24.02 -2.41 1 .21

26.61 23.65 -2.96 0.94

72 29.37 24.23 -5.14 3.94

29.80 24.31 -5.49 3.47

MOPS pH 5 0 25.22 23.62 -1 .60 0

25.14 23.48 -1 .66 0

2 24.13 23.83 -0.30 -1 .30

24.53 23.78 -0.75 -0.91 24 25.46 23.35 -2.1 1 0.51

24.88 23.58 -1.30 -0.36

72 26.28 23.38 -2.90 1.30

26.12 23.56 -2.56 0.90

The C _T values for c-fos and IL-β1 as well as for 18 S rRNA at time t = 0 h are comparable for both buffers. This shows that the use of the MOPS buffer did not introduce or retain any qPCR inhibitors into the sample during the work-up or the substances used in the buffers themselves acted as inhibitors.

The c-fos level of transcription of the EDTA-stored samples initially dropped sharply with a storage time of up to 24 h (AAC-r values up to -5.55), which suggests a reduction of the RNA increased with longer storage time but then probably strongly due to an ex vivo gene induction again. In the MOPS-stabilized samples, however, the c-fos transcription level dropped significantly weaker, and a significant ex vivo gene induction was not observed even in a period of 24 h.

Also with respect to the IL-1 ß transcription level was observed in the EDTA-stored samples, a significant increase in the AAC-r values over time, while the transcription level in the MOPS-stabilized samples even after 72 h in clear had increased to a lesser extent.

Example 3: Microscopic examination of the cell material in whole blood samples after stabilization

2.5 ml of blood were mixed with 5 ml of various stabilizing buffer and incubated for 24 h at room temperature. The blood samples, one of which a) was not mixed with a stabilizing buffer or one b) in one C) in the base buffer (MOPS, pH 5), d) in a 1, 2-dimethoxyethane-containing buffer (buffer 1 in Table 1), e) a sodium salicylate-containing buffer (Buffer 2), f) a glucosamine hydrochloride-containing buffer (Buffer 4) and g) a buffer containing sodium salicylate and glucosamine hydrochloride (Buffer 8) were incubated, then centrifuged for 5 min at 1000 xg, and the supernatant was up to 2.5 decanted ml. In order to assess the quality of the cell material, the samples were then examined under the microscope. When the cells were observed under the microscope, it could be seen that the cells which had been stored in the buffers according to the invention were still intact even after storage for 24 h at RT, while the unstabilized cells no longer represented the original cell morphology to any appreciable extent corresponded and were partially lysed. In particular, in the buffers b) and c), e) and g) even intact granulocytes were detected.

Example 4: Lysis of the cells and gel electrophoretic examination of the RNA

Further, as in Example 3 for 24 h stored in various stabilization buffers according to Table 1 samples were lysed according to the known protocols using the QIAzol lysis reagent. The RNA contained in the lysed sample was isolated by means of a phenol / chloroform extraction and purified by means of a RNeasy kit (Qiagen). Subsequently, the purified RNA was examined by means of a denaturing agarose gel after staining with SYBR Green. All examinations were performed twice.

The results of gel electrophoresis are shown in Figure 5. On the far left is shown an image of the electrophoresis gel of the RNA isolated from a blood sample unstabilized at room temperature for 24 h, while the RNA examined in gels II to V was stored in stabilizing buffers according to the invention (II: base buffer; III: buffer 2, pH IV; buffer 4, pH5; V: buffer 8). While the RNA obtained from the unstabilized stored blood sample in the gel is only weakly recognizable and also the intensity If the ratio of the 28 S rRNA band to the 18 S rRNA band is already clearly smaller than 2: 1, then both bands are clearly recognizable in the gels of the RNA obtained from the stabilized samples in a signal ratio of 28 S: 18 S of about 2: 1.

Example 5: Fluorescence-activated cell sorting (FACS) of the stabilized cells

Each 2.5 ml of blood were mixed with 5 ml of the different stabilizing buffer according to Table 1 and incubated for 24 h at room temperature. The cells contained in the sample were then labeled by means of a CD3-FITC antibody according to a standard protocol (incubation of the sample with 10-20% by volume CD3-FITC, incubation of the mixture at room temperature in the dark, erythrocyte lysis, centrifugation, washing with PBS Buffer, recentrifuging, adding the sample with 1% paraformaldehyde solution in PBS) and using a

FACSCalibur from BD Biosciences (San Jose, California, USA) was analyzed by flow cytometry.

In particular, buffer solutions of pH 5 containing (i) MOPS (base buffer), (ii) MOPS and 250 mg / ml sodium salicylate (buffer 2), (iii) MOPS and 100 mg / ml glucosamine hydrochloride (buffer 4 ) and (iv) 10% by volume of 1,2-dimethoxyethane in an MOPS-containing buffer (buffer 1) is very well suited for stabilizing the whole blood samples at room temperature while preserving the cell morphology, so that a cell-specific labeling of the cells remains after storage of the sample Cells with antibodies and subsequent fluorescence-activated cell sorting was possible. This is shown in Figure 6 by comparing a non-stabilized sample (a) stored in a citrate buffer at 4 ° C for 24 h with a sample incubated in the glucosamine hydrochloride-containing MOPS buffer for 24 h (b). clarified. On the left side, the dot plots of the blood samples before marking with CD3-FITC obtained by means of FACS analysis and in the middle the dot plots of the CD3-F ITC-labeled blood samples which have the pattern typical for leucocytes are shown. It can be seen from this that the system according to the invention does not lyse the cells. The pictures on the right show the for the samples a) and b) obtained histograms after labeling the leukocytes with CD3-FITC. It can be clearly seen that the cells stored in the buffer according to the invention are intact, so that they can stain with specific antibodies and be examined by FACS analysis. X-Axis: Forward Scatter (FSC) Y-Axis: Sideward Scatter (Side Scattered, SSC)

Claims

claims

1 . Use of an aqueous system containing one or more substances selected from the group consisting of 3 - (/ V-morpholino) propane sulfonic acid (MOPS), 1, 2-dimethoxyethane, sodium salicylate, hexaammonium heptamolybdate, glucosamine hydrochloride, indole, 2- ( 4-hydroxyphenyl) ethanol and tetrahexylammonium chloride, for the stabilization of nucleic acids in cell-containing biological samples to obtain the cell morphology of the cell material.

2. Use according to claim 1, wherein the aqueous system is MOPS or a mixture of MOPS and at least one further substance selected from the group comprising 1, 2-dimethoxyethane, sodium salicylate, hexaammonium heptamolybdate, glucosamine hydrochloride, indole, 2- (4-hydroxy - phenyl) ethanol and tetrahexylammonium chloride.

Use according to claim 2, wherein the aqueous system comprises MOPS or a mixture of MOPS with sodium salicylate and / or glucosamine hydrochloride.

Use according to any one of claims 1 to 3, wherein the aqueous system comprises at least one further substance selected from the group consisting of anticoagulants and organic solvents.

5. Use according to any one of claims 1 to 4, wherein the substances present in the aqueous system in a concentration range of 1 to 3000 mg / ml, preferably 25 to 2000 mg / ml, more preferably 50 to 1500 mg / ml and in particular 400 to 850 mg / ml.

Use according to any one of claims 1 to 5, wherein the aqueous system is a buffer and the pH of the buffer is 3 to 7, preferably 4 to 6 and more preferably 4.5 to 5.5.

Use according to any one of claims 1 to 6, wherein the biological sample comprises blood, preferably whole blood.

Use according to any one of claims 1 to 7, wherein the nucleic acids are ribonucleic acids (RNA).

9. A method for stabilizing nucleic acids in cell-containing biological samples to obtain the cell morphology of the cell material to provide samples for at least one of the following methods for assaying the nucleic acids contained in the sample: PCR, RT-PCR, electrophoretic methods, microarray analyzes , Marking, isolation and / or

Detection of the nucleic acids, comprising the displacement of the biological sample with a nucleic acid-stabilizing aqueous system according to any one of claims 1 to 8.

10. The method according to claim 9, comprising a step of immunohistological labeling of individual cell types in a sample containing different cell types prior to examination of those contained in the sample

Nucleic acids.

11. The method according to claim 9 or 10, wherein the labeling of the cell types is carried out with fluorescently labeled antibodies.

12. The method according to any one of claims 9 to 1 1, comprising a

Step for the selection and separation of individual cell types from a sample containing different cell types before the examination of the nucleic acids contained in the sample.

13. The method according to any one of claims 9 to 12, wherein the selection and separation of the cell types by means of fluorescence-based flow cytometry.

14. A kit for stabilizing, isolating or stabilizing and isolating nucleic acids from cell material-containing samples, comprising at least one aqueous system according to one of claims 1 to 8.