WO2017198715A1

WO2017198715A1 - Nucleic acid stabilization agent

Info

Publication number: WO2017198715A1
Application number: PCT/EP2017/061848
Authority: WO
Inventors: Robert Loewe
Original assignee: R Biopharm AG
Current assignee: R Biopharm AG
Priority date: 2016-05-17
Filing date: 2017-05-17
Publication date: 2017-11-23
Anticipated expiration: 2018-11-17

Abstract

The invention relates to a method, kit and sample collection container for stabilizing nucleic acids in a sample. The invention is based on treating a biological sample, such as body fluids, in particular blood, urine, sputum, or other samples containing biological cells or obtained from a mammalian organism, with one or more salts, comprising at least one salt according to the general Formula I, preferably comprising treating a sample with ammonium formate. The invention further relates to a method for the detection and/or isolation of a nucleic acid from a sample including, or subsequent to, the method for stabilizing said nucleic acids according to the invention, in particular wherein said sample comprises the salts of the invention after having been treated using the method of the invention. The invention also provides a kit for the detection, isolation and/or stabilization of nucleic acids from a biological sample and a collection container configured for the isolation of a biological, preferably blood sample.

Description

NUCLEIC ACID STABILIZATION AGENT

DESCRIPTION

BACKGROUND OF THE INVENTION

The stabilization of nucleic acids in biological samples is increasingly important in biological, medical and pharmacological research and diagnostics. Analysis of nucleic acids obtained from a biological sample enables one to determine the genetic characteristics of a sample or draw functionally relevant conclusions on the sample or on the cells within the sample.

For example, the analysis of ribonucleic acids (RNA), especially messenger RNA (mRNA), allows an accurate determination of levels of gene activity by means of gene expression analysis. Gene expression is now commonly used in methods of diagnostic and analytic importance, providing insights into cellular identity, disease state or other functional characteristics of a cell or subject.

The analysis of deoxyribonucleic acids (DNA) via methods such as PCR or DNA sequencing enables determination of genetic characteristics of a cell, or sample, or of the subject from which the sample is obtained. At present, many techniques are being developed for disease diagnosis, prognosis and risk prediction via analysis of genotype, for example by detecting single nucleotide polymorphisms (SNPs) or other alleles on a sequence-specific basis. Furthermore, the analysis of nucleic acids enables identification of infectious pathogens such as viruses, bacteria, etc. in the sample of a subject.

A key requirement for nucleic acid isolation and analysis is a rapid stabilization of nucleic acids immediately after the collection of a biological sample from its natural environment. Nucleic acids are subject to constant turnover in biological samples. Labile RNA molecules are degraded enzymatically, such that subsequent RNA separation and analysis is plagued by short life times of RNA molecules present in biological samples as well as high fragmentation of isolated RNA.

Upon collecting of a sample, ex vivo gene induction and down-regulation, initiation of cell death programs, and enzymatic activity can all contribute to changes in cellular RNA transcript levels and levels of other nucleic acid species. Furthermore, mechanical irritation during sample collection and subsequent transport or changes of physical conditions such as temperature changes during storage or transport cause the induction of gene transcription or nucleic acid degradation with the concomitant over- or underproduction of certain nucleic acid species.

To minimize experimental noise and increase reproducibility it is necessary to optimize the procedures of sample collection and extraction of nucleic acids. The increasing application of RNA and DNA analysis from samples of blood and other body fluids has led to an accelerating demand for potent and cost effective DNA and RNA stabilizing agents. Agents that stabilize nucleic acids in biological samples over several days at room temperature would simplify shipment and storage of such samples before nucleic acid extraction and analysis, as they could be shipped or stored at room temperature instead of processing the samples immediately or freezing them to prevent changes of the nucleic acid content after sample collection.

To date there are only a limited number of products available for effective nucleic acid stabilization, such as PAXgene (Qiagen), which exhibit high prices per tube or sample.

Furthermore, many of these products are specific to certain nucleic acid species such as RNA or DNA. At present, few universal stabilizing agents for nucleic acids in general have been successfully established on the market.

Approaches for stabilizing nucleic acids have been disclosed in the art. WO 2012018638 A2 describes chemical compounds, with which biological samples can be treated, thereby reducing nucleic acid degradation, comprising preferably the administration of a precipitating agent, at least one lower alcohol and at least one chaotrope together with the compounds described therein. WO 201 1 151427 A1 describes the use of a combination of chemical compounds in an aqueous buffer system that enables nucleic acid stabilization. Both methods require however multiple chemical components in an aqueous system at particular concentration ranges in order to achieve stabilization. WO 2013/024072 A1 and US 2002/146677 A1 describe methods for RNA stabilization comprising quarterney ammonium salts. US 2001/016312 A1 and WO 2015/1 10645 A1 describe nucleic acid stabilization using salts of organic acids. US 2008/248559 A1 describes a method for purification of RNA involving the use of formic acid. US 6 776 959 B1 describes a device for drawing blood containing a aqueous solution for nucleic acid stabilization. US

2013/137586 A1 discloses a method for stabilizing cells.

In light of the prior art, there remains a significant need to provide additional means for effective stabilization and nucleic acids in biological samples, such as in body fluids, blood, urine, sputum, and cerebrospinal fluid (CSF).

SUMMARY OF THE INVENTION

In light of the prior art the technical problem underlying the present invention is to provide alternative and/or improved means for stabilizing nucleic acids in a sample.

This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims. The invention therefore relates to a method for stabilizing nucleic acids in a sample, comprising treating said sample with one or more salts, comprising at least one salt according to Formula I:

wherein R is H or an alkyl group comprising 1-6 C atoms and X⁺ is a cation.

It was entirely surprising that the nucleic acids of a sample are stabilized by addition of a salt according to Formula I to the sample. The method of the present invention enables an effective, cost efficient and universal stabilization of nucleic acids in samples at room temperature for several days without significant changes in the nucleic acid identity or content within the sample. According to the knowledge of the inventor, no mention has been made in the art of using a salt comprising a carboxylate anion to treat a biological sample for enhanced nucleic acid stability.

The carboxylate anion of the salt employed in the present invention may also be described such that the negative charge that is left after deprotonation of the carboxyl group is delocalized between the two electronegative oxygen atoms in a resonance structure, such that Formula I may also be represented alternatively as:

As shown in more detail below, salts according to formula I are advantageous over known nucleic acid stabilizing agents as they stabilize all kinds of nucleic acids including various forms of RNA and DNA. Additionally, salts according to formula I stabilize nucleic acids over long periods of time as compared to known stabilizing agents, allowing longer storage of stabilized samples at RT before analysis of the nucleic acid content.

According to initial findings, the cells of the biological sample are not disrupted by the treatment described herein, for example with ammonium formate. It appears as though the cells are fixed in toto and can potentially be sorted, if required. Ammonium formate itself does not appear to cause cell lysis. This is particularly advantageous due to the possibility of conducting cell sorting techniques, such as FACS, or other cell-type isolating procedures, after stabilization of the nucleic acids within the cells. The sample can therefore be treated, the nucleic acids stabilized, then the cells sorted into sub-populations and these sub-populations from a sample then analysed for particular attributes of the nucleic acids, in particular RNA, comprised within.

In a preferred embodiment the method for stabilizing nucleic acids comprises:

Providing or obtaining a biological sample from a subject comprising nucleic acids, and Treating said sample with one or more salts, comprising at least one salt according to Formula I:

wherein R is H or an alkyl group comprising 1-6 C atoms and X⁺ is a cation, and preferably mixing or agitating said sample and/or preferably storage of said sample.

In a preferred embodiment the invention relates to a method for stabilizing nucleic acids in a sample, comprising treating said sample with one or more salts comprising at least a formic acid salt (formate), or salt with a formate anion, and an ammonium salt, wherein the ammonium salt comprises ammonium (NH₄ ⁺), or a primary, secondary or tertiary ammonium cation. Ammonium formate may be applied, or two or more different salts comprising formate anions and ammonium cations, wherein the ammonium cations are ammonium (NH₄ ⁺), or a primary, secondary or tertiary ammonium cation.

The anions of the salt according to formula I relate in general to carboxylate anions, in particular to anions according to Formula I wherein R is H (thereby a formate anion) or other alkyl group, preferably comprising 1 to 6 carbon atoms. Encompassed by the invention is the use of salts comprising formate, acetate, propionate, butyrate, isobutyrate (2-Methylpropanoate), pentanoate (valerate), isovalerate (3-Methylbutanoate) or hexanoate anions.

Formate is the conjugate base or anion derived from formic acid and is the simplest of the carboxylate anions. Examples of formate salts, without being limited thereto, are ammonium formate, calcium formate or potassium formate. Acetate is the conjugate base or anion derived from acetic acid. Examples of acetate salts, without being limited thereto, are ammonium acetate and sodium acetate. Propionate is the conjugate base or anion derived from propionic acid. Examples of propionate salts, without being limited thereto, are ammonium propionate, calcium propionate or sodium propionate. Butyrate (also known as butanoate) is the conjugate base or anion derived from butyric acid (also known as butanoic acid). Likewise, the carboxylate anions of C1-C6 are known to a skilled person and represent the conjugate base or anion of the corresponding acid. A skilled person is aware of salts comprising such anions.

According to Formula I, X⁺ is a cation. The selection of a suitable cation for salt formation with a carboxylate anion as described herein is within the ability of a skilled person. Numerous salts are available comprising a carboxylate anion and any given cation.

In a preferred embodiment of the invention at least one of the salts used for treating the sample contains an ammonium cation. The salt comprising an ammonium cation may be the same salt, or a different salt, than the salt comprising the carboxylate anions as described herein.

The cation may also relate to any inorganic cation such as but not limited to Sodium, Potassium, Calcium or Magnesium. The desired technical effect of nucleic acid stabilization is achieved upon obtaining the presence, in a functionally sufficient amount, of a carboxylate anion in solution in the sample. In a preferred embodiment the sample, in solution, also comprises an ammonium cation in a functionally sufficient amount to complement and preferably enhance the effect obtained by the carboxylate anion. In a preferred embodiment the carboxylate anion and ammonium cation are present in the same salt, such as in ammonium formate, but may be present in separate salts. In a preferred embodiment, the carboxylate anion, preferably of Formula I, provides a synergistic improvement in nucleic acid stabilization when combined with an ammonium cation, in particular a synergistic effect is achieved with a formate anion and an ammonium cation, preferably ammonium, primary ammonium cations, secondary ammonium cations and tertiary ammonium cations.

Ammonium cations include ammonium, primary ammonium cations, secondary ammonium cations, tertiary ammonium cations, and quaternary ammonium cations. Preferred are

ammonium, primary ammonium cations, secondary ammonium cations and tertiary ammonium cations.

Primary ammonium cations relate to NH3+R, wherein R is alkyl or aryl. Secondary ammonium cations relate to NH2+R1 R2, wherein R1 and R2 may be the same or different and are alkyl or aryl. Tertiary ammonium cations relate to NH+R1 R2R3, wherein R1 , R2 and R3 may be the same or different and are alkyl or aryl. Quaternary ammonium cations, also known as quats, are positively charged polyatomic ions of the structure NR4+, R being an alkyl group or an aryl group.

Preferred embodiments of ammonium cations are selected from the group consisting of ammonium, Tetramethylammonium ((CH3)4N), Triethylammonium ((CH3CH2)3NH+), Met-Lys (C1 1 H23N303S) and Pyridinium (C5H5N).

Primary ammonium cations comprise, but are not limited to, methylammonium and

ethylammmonium.

Secondary ammonium cations comprise, but are not limited to, dimethylammonium,

diethylammonium and Met-Lys (dipeptide formed from L-methionine and L-lysine residues).

Tertiary ammonium cations comprise, but are not limited to, trimethylammonium and

triethylammonium.

In a preferred embodiment the one or more salts to be employed (or at least the cation and anion of two separate salts to be employed) comprises the formiate anion and an ammonium cation selected from the group consisting of ammonium, primary ammonium cations, secondary ammonium cations and tertiary ammonium cations, wherein the primary ammonium cations relate to NH3+R, wherein R is alkyl or aryl, wherein the secondary ammonium cations relate to

NH2+R1 R2, wherein R1 and R2 may be the same or different and are alkyl or aryl, wherein tertiary ammonium cations relate to NH+R1 R2R3, wherein R1 , R2 and R3 may be the same or different and are alkyl or aryl, wherein preferred embodiments of the ammonium cations are selected from the group consisting of ammonium, Triethylammonium ((CH3CH2)3NH+), Met-Lys (C1 1 H23N303S) and Pyridinium (C5H5N), preferred embodiments of primary ammonium cations comprise, but are not limited to, methylammonium and ethylammonium, preferred embodiments of secondary ammonium cations comprise, but are not limited to, dimethylammonium, diethylammonium and Met-Lys (dipeptide formed from L-methionine and L-lysine residues), and preferred embodiments of tertiary ammonium cations comprise, but are not limited to, trimethylammonium and triethylammonium.

In a preferred embodiment the one or more salts to be employed (or at least the cation and anion of two separate salts to be employed) comprises the acetate anion and an ammonium cation selected from the group consisting of ammonium, primary ammonium cations, secondary ammonium cations and tertiary ammonium cations, wherein the primary ammonium cations relate to NH3+R, wherein R is alkyl or aryl, wherein the secondary ammonium cations relate to

NH2+R1 R2, wherein R1 and R2 may be the same or different and are alkyl or aryl, wherein tertiary ammonium cations relate to NH+R1 R2R3, wherein R1 , R2 and R3 may be the same or different and are alkyl or aryl, wherein preferred embodiments of ammonium cations are selected from the group consisting of ammonium, Triethylammonium ((CH3CH2)3NH+), Met-Lys

(C1 1 H23N303S) and Pyridinium (C5H5N), preferred embodiments of primary ammonium cations comprise, but are not limited to, methylammonium and ethylammonium, preferred embodiments of secondary ammonium cations comprise, but are not limited to, dimethylammonium, diethylammonium and Met-Lys (dipeptide formed from L-methionine and L-lysine residues), and preferred embodiments of tertiary ammonium cations comprise, but are not limited to, trimethylammonium and triethylammonium.

In a preferred embodiment the one or more salts to be employed (or at least the cation and anion of two separate salts to be employed) comprises the propionate anion and an ammonium cation selected from the group consisting of ammonium, primary ammonium cations, secondary ammonium cations and tertiary ammonium cations, wherein the primary ammonium cations relate to NH3+R, wherein R is alkyl or aryl, wherein the secondary ammonium cations relate to

In a preferred embodiment the one or more salts to be employed (or at least the cation and anion of two separate salts to be employed) comprises the butyrate anion and an ammonium cation selected from the group consisting of ammonium, primary ammonium cations, secondary ammonium cations and tertiary ammonium cations, wherein the primary ammonium cations relate to NH3+R, wherein R is alkyl or aryl, wherein the secondary ammonium cations relate to

In a preferred embodiment the one or more salts to be employed (or at least the cation and anion of two separate salts to be employed) comprises the isobutyrate anion and an ammonium cation selected from the group consisting of ammonium, primary ammonium cations, secondary ammonium cations and tertiary ammonium cations, wherein the primary ammonium cations relate to NH3+R, wherein R is alkyl or aryl, wherein the secondary ammonium cations relate to

In a preferred embodiment the one or more salts to be employed (or at least the cation and anion of two separate salts to be employed) comprises the isovalerate anion and an ammonium cation selected from the group consisting of ammonium, primary ammonium cations, secondary ammonium cations and tertiary ammonium cations, wherein the primary ammonium cations relate to NH3+R, wherein R is alkyl or aryl, wherein the secondary ammonium cations relate to

In a preferred embodiment the one or more salts to be employed (or at least the cation and anion of two separate salts to be employed) comprises the hexanoate anion and an ammonium cation selected from the group consisting of ammonium, primary ammonium cations, secondary ammonium cations and tertiary ammonium cations, wherein the primary ammonium cations relate to NH3+R, wherein R is alkyl or aryl, wherein the secondary ammonium cations relate to NH2+R1 R2, wherein R1 and R2 may be the same or different and are alkyl or aryl, wherein tertiary ammonium cations relate to NH+R1 R2R3, wherein R1 , R2 and R3 may be the same or different and are alkyl or aryl, wherein preferred embodiments of ammonium cations are selected from the group consisting of ammonium, Triethylammonium ((CH3CH2)3NH+), Met-Lys

In a preferred embodiment the one or more salts to be employed (or at least the cation and anion of two separate salts to be employed) does not comprise the ammonium acetate.

In a preferred embodiment the invention relates to a method for stabilizing nucleic acids in a sample, comprising treating said sample with one or more salts comprising at least a formic acid salt (formate) and an ammonium salt, wherein the ammonium salt comprises ammonium (NH₄ ⁺), or a primary, secondary or tertiary ammonium cation.

Ammonium is the preferred embodiment of this group of cations. Different ammonium cations have different characteristics in terms of solubility and lipophilic properties due to the length, number and nature of their organic side chains. This can be used to adjust the properties of the salt to the characteristics of a specific sample, in which nucleic acids should be stabilized.

In a preferred embodiment of the invention the sample is treated with at least a formic acid salt (formate) and an ammonium salt (salt comprising an ammonium cation). Formic acid salts and ammonium salts are typically of low cost and show good solubility in aqueous solutions, which is advantageous for stabilization of liquid biological samples. Examples of ammonium salts are, but are not limited to, ammonium formate, ammonium lactate, ammonium carbonate, ammonium chloride, ammonium acetate, ammonium propionate, ammonium isobutyrate, ammonium pentanoate, ammonium isovalerate (3-Methylbutanoate), ammonium hexanoate and ammonium butyrate.

In another preferred embodiment of the invention the salt is ammonium formate, which can be provided in its crystalline solid form or in an aqueous solution, such as in a buffer. It can be provided in liquid or solid form in a collection container configured for collection and/or storage of the sample containing nucleic acids to be stabilized. The finding that ammonium formate, preferably in the concentrations in solution in the sample as disclosed herein, could lead to stabilization of nucleic acids in a sample represents an unexpected and beneficial finding that was, to the knowledge of the inventors, not previously suggested in the art.

In a preferred embodiment of the invention the nucleic acid to be stabilized is RNA and/or the sample for stabilization comprises RNA. RNA content of biological samples is highly instable due to degradation and selective RNA synthesis after sample collection. RNA degradation due to hydrolysis by RNases is a crucial problem if samples are stored at room temperature before processing. Stabilization of RNA by the salts according to Formula I and specifically ammonium formate was entirely surprising and is a cost efficient and simple way of stabilizing the RNA content of a sample, thereby avoiding time and cost intensive storage by freezing or refrigeration. In a preferred embodiment the RNA to be stabilized is mRNA, in particular mRNA in a whole blood sample, or in sample derived from blood, such as plasma or serum.

In another preferred embodiment of the invention the nucleic acid to be stabilized is DNA and/or the sample for stabilization comprises DNA. The DNA content of biological samples can change after sample collection for example due to cell division, DNA synthesis or DNA degradation in apoptotic or necrotic cells. Stabilization of DNA content of a sample by combining the sample with a salt according to formula I or ammonium formate will facilitate handling of samples whose DNA content should be analysed.

In one embodiment the sample can be any sample comprising a nucleic acid, either in aqueous or in dried form, such as in freeze-dried form. Biological samples may be diluted or treated in advance, suh that the sample is not necessarily to be characterized as a biological sample. In a preferred embodiment of the invention the sample is a biological sample obtained from, or comprising, one or more biological cells, organisms or viruses, a bodily fluid of a mammal, such as blood, urine, sputum, cerebrospinal fluid (CSF), a body swab, body smear, or a tissue sample, preferably obtained from a mammalian subject. Stabilization of nucleic acid content of such samples will facilitate handling and analysis of such samples for diagnostics, analytic or research purposes.

In another preferred embodiment of the invention the sample is a blood sample or derived from a blood sample, such as whole blood, plasma or serum. Stabilization of the nucleic acid content of such samples is specifically useful as serum contains DNases and lysis of blood cells during collection and storage can lead to release of RNases leading to changes of the nucleic acid content of such samples, if not stabilized. Blood samples are of particular relevance for the diagnostic industry and have proven to date difficult sample types with respect to stabilizing effectively all the nucleic acid material within the sample. Blood samples are typically complex and protein rich, thereby complicating standard stabilization and isolation procedures.

Surprisingly, the method and means of the present invention enable effective stabilization of nucleic acids in a blood sample regardless of blood cell presence or protein content.

A stabilized whole blood sample using the present invention shows remarkably lengthy times for which the nucleic acids within the sample are stable/maintain integrity. A blood sample can be stored and transported at a temperature lower than room temperature, for example at 2°C to 8°C, or at temperatures of room temperature at approximately 20°C without significant reduction in nucleic acid integrity.

The stabilized whole blood sample can also be stored under conditions where it is frozen, although this is not necessary for maintaining nucleic acid integrity over times of 1 -6 days. RNA integrity in a blood sample is typically maintained for approximately 6 days when stored at 20°C. For longer storage and archiving, the samples can be stored at 2°C to 6°C for about 20 days, and at about -20°C for around 3 months, thereby maintaining RNA integrity in a blood sample. For later nucleic acid isolation, the sample can be thawed and further processed, if necessary. It has been found that the stabilizing salts described herein allow reliable freezing of blood samples for later RNA isolation.

In a preferred embodiment of the invention the treatment of the sample with one or more salts leads to a formate concentration of 0.01 M to 10 M, preferably 0.05 M to 8 M, more preferably 0.1 M to 7 M, or 0.25 M to 6 M in said sample. It was surprising that the salts employed were effective over such a broad concentration range, thereby allowing for effective stabilization and high flexibility in terms of sample volume to be added to a collection container containing the stabilizing salt. Methods for determining formate concentration in solution are known to a skilled person, for example the Formate Assay Kit from Sigma Aldrich, Catalog Number: MAK059.

In a preferred embodiment of the invention the treatment of the sample with one or more salts leads to an ammonium concentration of 0.01 M to 10 M, preferably 0.05 M to 8 M, more preferably 0.1 M to 7 M, or 0.25 M to 6 M in said sample. This range of ammonium concentration allows for high flexibility in terms of sample volume to be added to a collection container containing the stabilizing salt. Methods for determining ammonium concentration in solution are known to a skilled person, for example the Ammonia Assay Kit from Sigma Aldrich, Catalog Number: AA0100.

These concentrations, namely of 0.01 M to 10 M, preferably 0.05 M to 8 M, more preferably 0.1 M to 7 M, or 0.25 M to 6 M in said sample, also apply to the other carboxylate anions and to the other ammonium cations as described herein.

In a preferred embodiment of the invention the sample is treated with an ammonium formate stock solution, preferably at a concentration of 1 - 20 M. This concentration range of an ammonium formate solution allows for high flexibility in terms of combining different volumes of sample and stabilizing solution to be combined.

In another preferred embodiment of the invention the sample is treated with ammonium formate in a solid form, for example in crystalline form, which will allow for longer storage life of collection containers comprising ammonium formate configured for the collection and stabilization of nucleic acids.

A further aspect of the invention relates to a method for the detection and/or isolation of a nucleic acid from a sample, conducted in combination with, preferably subsequently to, the method for stabilization described above.

In a preferred embodiment the method comprises:

providing or obtaining a sample, preferably biological sample from a subject, comprising nucleic acids, and

treating said sample with one or more salts, comprising at least one salt according to

Formula I:

o wherein R is H or an alkyl group comprising 1-6 C atoms and X⁺ is a cation, and preferably mixing or agitating said sample and/or storage of said sample, and

isolating and/or detecting said stabilized nucleic acids from the sample.

A further aspect of the invention relates to a method for the detection and/or isolation of a nucleic acid from a sample, wherein said sample comprises a formate concentration of 0.01 to 10 M and/or an ammonium concentration of 0.01 M to 10 M.

In a preferred embodiment the method comprises:

providing or obtaining a sample, preferably biological sample from a subject, comprising nucleic acids,

treating said sample with one or more salts, such that a formate concentration of 0.01 to 10 M and/or an ammonium concentration of 0.01 M to 10 M in said sample is achieved, and

preferably mixing or agitating said sample and/or storage of said sample, and

isolating and/or detecting said stabilized nucleic acids from the sample.

The present invention therefore also encompasses methods of detecting and/or isolating nucleic acids from samples having being previously treated with the salts as described in the method of the present invention. The stabilizing step via treatment of the salts is not necessarily an essential feature of the invention, for example in embodiments where nucleic acids are isolated and/or detected from samples by separate parties to those who have obtained, processed and/or stabilized the sample using a method corresponding to the invention disclosed herein. The invention therefore directly encompasses the subsequent isolation and/or detection of nucleic acids from samples comprising the salts as described herein.

Methods of detecting the stabilized nucleic acids are known to a skilled person, including methods such as but not limited to the polymerase chain reaction (PCR) or DNA sequencing.

Methods of detecting the stabilized nucleic acids are preferably related to quantitative or semiquantitative nucleic acid techniques, such as, but not limited to, qPCR, RT-PCR, Next Generation Sequencing (DNA/RNA-Sequencing), DNA/RNA- in situ hybridization, enzymatic restriction assay, RNase protection assay, ligation assay, microarrays, protein binding assay, aptamer binding assay, pyro-sequencing, Sanger sequencing or isothermal amplification.

Detection of the nucleic acid may be conducted directly on the stabilized sample. Surprisingly, the salts of the present invention do not interfere with PCR or other enzymatic nucleic acid detection or conversion techniques. The salts employed herein do not interfere with reverse transcription, thereby enabling cDNA production from mRNA in a biological sample. Stabilization of the nucleic acid content of a sample by introducing the salts described herein, preferably ammonium formate, in the sample allows for generation of reliable and reproducible data from analytical nucleic acid techniques independent of storage time or temperature of the sample.

Alternatively, the sample may be subsequently treated to isolate the stabilized nucleic acids. Methods for isolating nucleic acids are known to a person skilled in the art of analytical chemistry, biochemistry or molecular biology. Such methods include, but are not limited to those described in Sambrook J et al. (1989). Molecular Cloning: A Laboratory Manual. (New York: Cold Spring Harbor Press), or in Nucleic Acid Isolation and Purification, 4^th Edition, from Roche

(https://lifescience.roche.com). As described in the BioRad guide to Nucleic Acid Extraction and Purification (www.bio-rad.com), nucleic acid sample preparation involves a range of techniques to transform a sample which cannot be directly analyzed into one that fits the requirements of the analytical technique to be used, in some cases such as reverse transcription (RT) or PCR.

When working with cells or tissues as the starting material, the first step of the nucleic acid isolation process is cell lysis or membrane permeabilization. This process breaks open the cell membranes and disrupts the cellular structure to create a cell lysate. This allows the nucleic acid of interest to be accessed and separated away from unwanted cellular components.

For example, RNA may be isolated by filter column purification can be used to purify RNA from mammalian cell cultures, bacteria, and yeast, as well as plant and animal tissue having been treated according to the present invention. By adjusting the pH and salt of the solution, RNA can be separated from the cellular debris or other contaminants by absorbing the RNA onto a silica membrane situated at the base of a filter column. An optional DNase digest can be performed to minimize residual genomic DNA contamination. The purified RNA is then eluted off the membrane with an elution buffer into a collection tube. RNA isolated using column purification is suitable for use in a variety of downstream applications including RT-PCR, real-time PCR, and northern blot analysis. Alterations of the column purification method can also be used to isolate small RNAs such as microRNAs. A variation of the phenol/chloroform DNA extraction method paired with guanidinium isothiocyanate can be used to obtain RNA of high purity.

Filter column purification can be used to purify DNA from mammalian cell cultures, bacteria, and yeast, as well as plant and animal tissue after treatment according to the present invention. By adjusting the pH and salt of the solution, DNA of interest can be separated from cellular debris or other unwanted contaminants by binding the DNA to a silica membrane situated at the bottom of a filter column. After the DNA is bound to the membrane, it is subjected to a low and a high stringency wash to remove contaminants such as RNA, proteins and lipids. The purified DNA is eluted off the membrane with an elution buffer into a collection tube. The volume of the elution buffer used can be varied depending on the final concentration of plasmid or DNA desired.

Recovered DNA is suitable for use with PCR and Southern blot analysis. A traditional DNA purification method that can be used to obtain highly pure DNA is phenol/chloroform extraction followed by ethanol precipitation. This method is intended for the extraction of DNA from animal and plant tissues, cultured mammalian cells, bacteria and yeast cells in under one hour. As an alternative to filter column purification or phenol/chloroform extraction, a buffered ion exchange resin can be used for the removal of PCR contaminates from blood, cultured cells, and bacteria.

A further aspect of the invention relates to a kit for the detection, isolation and/or stabilization of nucleic acids from a biological sample, comprising one or more salts, comprising at least one salt according to Formula I:

wherein R is H or an alkyl group comprising 1-6 C atoms and X⁺ is a cation, and additional means for nucleic acid isolation.

The means for nucleic acid isolation are selected from the group consisting of detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, such as guanidinium isothiocyanate, guanidinium hydrochloride, sodium dodecylsulfate, polyoxyethylene sorbitan monolaurate, RNAse inhibitor proteins, chloroform, isoamyl-alcohol, isopropanol, methanol, ethanol, glacial acetic acid, phenol and combinations thereof.

In a preferred embodiment of the invention the kit as described herein is characterised in that the kit comprises one or more salts, comprising at least one salt comprising a formate anion and at least one salt comprising an ammonium ion, preferably ammonium formate.

Such a kit combines simple sample collection with stabilization of the nucleic acid content, while subsequent nucleic acid isolation is facilitated by the provision of the means needed thereto. Such a kit may relate to those kits presently on the market or known for nucleic acid isolation, such as those comprising spin columns/filter columns for nucleic acid isolation.

The nucleic acids obtained from the method or kit can be subsequently further processed, for example reverse transcribed and amplified by means of RT-PCR methods in the case of RNA, amplified by means of PCR methods in the case of DNA and/or analyzed by means of electrophoretic methods such as Northern blotting (RNA) or Southern blotting (DNA) or microarrays. For the purposes of the invention, the term RT-PCR encompasses in particular so- called quantitative RT-PCR methods (qRT-PCR or real-time RT-PCR), which allow quantification of the mRNA obtained

A further aspect of the invention relates to a collection container configured for the isolation of a blood sample, wherein said container comprises one or more salts, comprising at least one salt according to Formula I:

wherein R is H or an alkyl group comprising 1-6 C atoms and X⁺ is a cation,

and wherein said container comprises:

a device for attachment of said container to a canula or syringe,

is a syringe suitable for blood isolation, exhibits an internal pressure less than atmospheric pressure, such as is suitable for drawing a pre-determined volume of sample into said container, and/or

comprises additionally detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, such as guanidinium isothiocyanate, guanidinium hydrochloride, sodium dodecylsulfate, polyoxyethylene sorbitan monolaurate, RNAse inhibitor proteins, and mixtures thereof, and/or

A filter system containing nitro-cellulose, silica matrix, ferromagnetic spheres, a cup retrieve spill over, trehalose, fructose, lactose, mannose, poly-ethylen-glycol, glycerol, EDTA, TRIS, limonene, xylene, benzoyl, phenol, mineral oil, anilin, pyrol, citrate, and mixtures thereof.

Such a container facilitates blood sample collection and nucleic acid stabilization immediately as it provides key structural features of the container required for this workflow.

The method, kit and container of the invention thereby enable introducing a biological sample immediately on collection into a container, which preferably comprises the stabilizing salt(s) as described herein. In preferred embodiments the biological sample is prepared and immediately introduced directly into the collection container. In preferred embodiments, the biological sample is withdrawn from the patient directly into the collection container without any intervening process or handling steps so that the sample mixes with the gene induction blocking agent immediately to prevent or inhibit nucleic acid decomposition. It has been found that collecting the biological sample directly from the patient, such as when collecting a whole blood sample, and introducing the sample directly into the container containing the stabilizing agent substantially prevents gene transcription and decomposition of the nucleic acids that otherwise occur when the sample is stored without the stabilizing agent.

The preferred volume of the container is between 0.10 mL and 50 ml_, preferably between 0.20 ml. and 10 mL. This preferred volume also relates to the sample volume of the method as described herein.

DETAILED DESCRIPTION OF THE INVENTION

All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.

The term "nucleic acids" refers to nucleic acid molecules including, without limitation, DNA, ssDNA, dsDNA, RNA, mRNA, tRNA, IncRNA, ncRNA, microRNA, siRNA, rRNA, sgRNA, piRNA, rmRNA, snRNA, snoRNA, scaRNA, gRNA or viral RNA.

For the purposes of the invention, any sample which contains biological cells, or was obtained from a mammalian organism, such as a bodily fluid, is referred to as a biological sample. The samples can, for example, be obtained 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, or from a lavage, from plants, plant parts and plant extracts, for example juices, fungi, prokaryotic or eukaryotic microorganisms such as bacteria or yeasts, fossil or mummified samples, soil samples, sludge, wastewaters and foodstuffs. Preferably, the biological sample comprises blood, particularly preferably whole blood. The term "sample" includes preferably, without being limitation, a bodily fluid, such as blood, urine, sputum, cerebrospinal fluid (CSF), a body swab, body smear, or a tissue and/or fluid sample obtained from a mammalian subject.

The term "treatment" encompasses essentially any method of contacting a sample with the agents described herein. Treatment may in some embodiments comprises bringing a solution comprising the stabilizing salts into contact with a liquid sample, bringing a solution comprising the stabilizing salts into contact with a solid sample (such as tissue), bringing a dried or crystalline form comprising the stabilizing salts into contact with a liquid sample, or other similar

embodiment. The treatment may comprise, but is not in all cases necessary, the agitation or mixing of the sample with the stabilizing agent. Methods for mixing or agitation are known to a skilled person.

The term "stabilizing" herein refers to treatment of a sample, which leads to conservation of the nucleic acid content present in the sample at the time of treatment. For the purposes of the invention, a sample is referred to as stabilized when the integrity of the contained nucleic acids after storage at a given temperature for a specified time is greater than the integrity of the nucleic acids in a (unstabilized) comparative sample which originates from the same source and was collected and stored under identical conditions, but without addition of a stabilization salts as described herein. The integrity of nucleic acids can be assessed by various techniques. Nucleic acids can be isolated and separated via gel electrophoresis, in order to provide information on the size and amount of nucleic acid molecules. Quantitative or semi-quantitative nucleic acid techniques may also be used in order to assess nucleic acid integrity, for example qPCR or RT- PCR may be employed to determine the amount of any given nucleic acid in a quantitative manner and to compare these values between multiple samples.

A salt is typically considered an ionic compound that results from the neutralization reaction of an acid and a base (Clugston M. and Fleming R. (2000). Advanced Chemistry (1 st ed.). Oxford: Oxford Publishing, p. 108.). Salts are composed of related numbers of cations (positively charged ions) and anions (negative ions) so that the product is electrically neutral (without a net charge).

The salts of the present invention are intended to dissolve in water, in particular in the aqueous biological samples in which nucleic acids are to be stabilized. Solution of the salts in the sample arises according to common principles, namely due to the salt's positive ions (e.g. ammonium) attracted the partially negative oxygen atom of H20, and the salt's negative ions (e.g. carboxylate anion) attract the partially positive hydrogens of H20. As such, in the preferred embodiment of employing a carboxylate anion and a cation of an ammonium salt, the carboxylate anion and cation of an ammonium salt must not be provided necessarily as a single ammonium salt (e. g. ammonium formate), rather the carboxylate anion and cation of an ammonium salt may arise from separate salts added to the sample.

A selection of appropriate salts for the required solubility and concentration in the biological sample lies within the skills of a chemist, biochemist or molecular biologist. Common salt-forming cations include Ammonium NH4+, Calcium Ca2+, Iron Fe2+, and Fe3+, Magnesium Mg2+, Potassium K+, primary, secondary, tertiary or quaternary ammonium NR4+, R being H, an alkyl group or an aryl group, or Sodium Na+. Common salt-forming anions include Acetate, Carbonate, Chloride, Citrate, Fluoride, Nitrate, Propionate, Butyrate, Isobutyrate, Isovalerate, Hexanoate, Phosphate or Sulfate.

A preferred embodiment the invention relates to the use of an "ammonium cation", which is defined as a positively charged polyatomic ion with the chemical formula NR1 R2R3R4+, which is formed by the protonation of ammonia (NH₃) or other positively charged substituted primary, secondary, tertiary or quaternary ammonium cations, wherein R1-R4 can be H, alkyl or aryl. The term "Ammonium cation" comprises, without being limited to, the molecules ammonium,

Tetramethylammonium ((CH₃)₄N), Triethylammonium((CH₃CH₂)₃NI-l⁺), Met-Lys (C11 H2₃N₃O₃S), and Pyridinium (C5H5N). Primary ammonium cations comprise, but are not limited to, methylammonium and ethylammmonium. Secondary ammonium cations comprise, but are not limited to, dimethylammonium, diethylammonium and Met-Lys (dipeptide formed from L- methionine and L-lysine residues). Tertiary ammonium cations comprise, but are not limited to, trimethylammonium and triethylammonium.

The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group of preferably 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, w-butyl, isobutyl, f-butyl, pentyl, hexyl and the like. Preferred alkyl groups have 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms.

The term "aryl" refers to any carbon-based aromatic group including, but not limited to, benzene, naphthalene, etc. The term "aromatic" also includes "heteroaryl group," which is defined as an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorous.

"Carboxyl" refers to a -COOH radical. Substituted carboxyl refers to -COOR where R is aliphatic, heteroaliphatic, alkyl, heteroalkyl, or a carboxylic acid or ester.

A functionally sufficient amount refers to any concentration of a salt as described herein suitable to achieve stabilization of the nucleic acids as described herein.

The term "kit" relates to a product, typically packaged in a single unit, or optionally in multiple units or packages, that comprises the components required for carrying out one or more of the methods described herein. The kit may contain the salts, tubes, filters, buffers, or instructions for carrying out a method, as is necessary for the end user. The kit of the present invention enables the combined packaging and provision of multiple components required in the method in an easy to use and compact format. The container of the present invention may also be provided in the kit as described herein.

Devices for attachment of a container to a cannula or syringe are known to a skilled person, and may incorporate fittings between tubing or container components to enable simple access to the patients' blood stream. A venous cannula is one inserted into a vein, primarily for the

administration of intravenous fluids, for obtaining blood samples and for administering medicines. Identifying fittings for specific cannula attachment between the container and the cannula are within the ability of a skilled person.

Detergents may also be present in the kit of the invention, and can be anionic detergents, cationic detergents or nonionic detergents. The anionic detergent can be, for example, sodium dodecyl sulfate. Nonionic detergents can be, for example, ethylene oxide condensation products, such as ethoxylated fatty acid esters of polyhydric alcohols. A preferred nonionic detergent is a polyoxyethylene sorbitan monolaurate sold under the trade name TWEEN 20 by Sigma Chemical Co. Another suitable detergent is sodium dodecylsulfate. The detergents are included in an effective amount to lyse the cells. Examples of chaotropic salts include urea, formaldehyde, guanidinium isothiocyanate, guanidinium hydrochloride, formamide, dimethylsulfoxide, ethylene glycol and tetrafluoroacetate. A suitable ribonuclease inhibitor is placental RNAse inhibitor protein.

FIGURES

The invention is further described by the following figures. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

In the following, the term PAXgene relates to the stabilization technology, method or solution according to WO 2013/024072 A1.

Brief description of the figures:

Figure 1 : Comparison of RNA stability of blood samples treated with PAXgene or ammonium formate.

Figure 2: Stabilization in ammonium formate maintains RNA quality.

Figure 3: RNA stabilization in multiple samples is reproducible.

Detailed description of the figures:

Figure 1 : Blood samples were stabilized in PAXgene (A) or ammonium formate (B-F) and were stored for the indicated time at 10°C. At the indicated time points, RNA was purified from the samples and transcribed into cDNA and expression of reference genes was measured by quantitative PCR.

(A) Blood samples were stabilized in PAXgene and stored for 0, 1 , 2 or 3 days at 18-25°C.

Expression of a reference gene was measured at each time point and fold changes in expression compared to day 0 are plotted. Red line indicates average of the reference gene. (B-F) 4 blood samples were stabilized in ammonium formate and stored for 0, 2, 5, 6, 13 or 14 days at 20°C. Two blood samples from the same patient were run in technical replicates in the PCR.

Expression of reference genes ACTB (C), C6orf64 (D), RPL13A (E) and RPL32 (F) were analyzed at each time point and average fold changes in expression as compared to day 0 are plotted for each sample in (B). Figure 2: MS2 RNA was added to urine samples. Subsequently samples were stabilized at room temperature for 48 or 72 hours. At the indicated time point, reverse transcription and qPCR with MS2 specific primers were performed on the samples, demonstrating that RNA quality was maintained after 72h as compared to 48 hours.

Figure 3: Multiple blood samples from different patients were collected and stabilized in ammonium formate and PAXgene and stored at 20 °C for 3 days. Samples were generated in the same facility simultaneously to represent a general output information in regards to standard procedure (to show that under routine conditions ammonium formate provides higher output of isolated RNA).

Subsequently, RNA was isolated from the samples and was transcribed to cDNA and relative expression of the PGK gene was determined by quantitative PCR. Each dot represents one sample. This shows that the quantity of RNA isolated after ammonium formate stabilization and isolation and post reverse transcription is higher when compared to the paxgene technology.

EXAMPLES

The invention is further described by the following examples. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

Methods employed in the Examples

Stabilization of blood with ammonium formate and total nucleic acid (TNA) isolation from ammonium formate stabilized blood

1 . Materials:

Eppendorf Research plus pipette 0,5-1 ΟμΙ.

Eppendorf Research plus pipette 10-1 ΟΟμΙ.

Eppendorf Research plus pipette 100-1000μΙ.

Eppendorf Thermomixer comfort 1 ,5ml / 2,0ml.

Eppendorf centrifuge 5810 R.

Eppendorf centrifuge 5424.

- Sarstedt Biosphere filter tips 0,5-20 μΙ; Ref.70.1 1 14.210.

- Sarstedt Biosphere filter tips 2-100 μΙ; Ref.70.760.212.

- Sarstedt Biosphere filter tips 1250 μΙ; Ref.70.1 186.210.

- Sarstedt Eppendorf filter tips 300 μΙ; Ref.022493004.

Sarstedt 50 ml reaction container; Ref.62.547.254.

- Sarstedt SafeSeal 1 ,5 ml reaction container; Ref.72.706.400.

Genaxxon DNA purification column Ref.S5379.0050.

- Satino Papiertuch Ref.163028.

- Proteinase K. Bioron, Cat.No.: 405010

- CaCI2 (1 M) RNase free water

- Ethanol 80%

Isopropanol 100%

2. Stabilization:

3,0 ml of whole blood were mixed with the 1 ,8 ml of ammonium formate (concentration = 10 M in Water) in a 50 ml sample container. Mixing occurred in the sample container after blood is drawn by inverting the tube 10 times or vigorous shaking. Subsequently, the reaction containers containing the samples were centrifuged at 3000x g for 10 minutes, which lead to a separation of the solution in the reaction containers into 3 phases:

The upper phase contained excess ammonium formate and serum like liquid.

The middle phase formed a viscous non dense pellet and contained cellular debris with low TNA content and represents a potential source for inhibitors.

The bottom phase formed a dense pellet containing the desired TNA.

The supernatant comprising the upper and the middle phase were discarded via decanting the liquid and remove all residual supernatant by draining the sample container on a paper towel.

3. Lysis:

To the resulting pellet 500 μΙ of lysis buffer (Guanidine hydrochloride, Triton X, Tris HCI, EDTA, DTT, PEG8000, CaCI2, Citric Acid, Proteinase K) was added and the pellet was resuspended in the lysis buffer by pipetting up and down. The resulting suspension/lysate was transferred to a 1 .5 ml reaction container and 100 μΙ of Proteinase K and 150 μ I of CaCI2 were added to the solution. The 1.5 ml reaction container was closed and the suspension was incubated at 56 °C for 45 minutes in a Thermomixer while vortexing at 1 .000 rotations per minute (rpm).

4. Binding:

After incubation at 56 °C for 45 minutes the 1 .5 ml sample container containing the lysate is briefly vortexed and subsequently centrifuged at 16.000x g for 3 minutes. The resulting supernatant is transferred without the pellet to a new 1.5 ml reaction container, which is again centrifuged at 16.000x g for 3 minutes. The resulting supernatant is transferred without the pellet to a new 1.5 ml reaction container and the volume of the lysate is determined by means of a Eppendorf Research plus pipette. Subsequently a defined volume of isopropanol (100 %) is added to the lysate. The volume of isopropanol is determined by dividing the volume of the lysate by 9.

Example: Volume lysate: 1200 μΙ

Volume isopropanol = 1200 μΙ : 9 = 133,3 μΙ

After adding the calculated volume of isopropanol absolute to the lysate, the 1 .5 ml reaction container was closed and mixed by vortexing. The 1.5 ml reaction container was centrifuged at 16.000x g for 3 minutes. The supernatant without pellet was transferred to an isolation column, which was placed in a suitable collection vessel. The assembled isolation column and the collection vessel were subsequently centrifuged at 6.000x g for 30 seconds. After centrifugation the column was carefully removed from the collection vessel and the flow through, which was collected in the collection vessel, was discarded.

5. Washing:

500 μΙ Ethanol (80%) were added to the isolation column and the column was placed in the collection vessel. The assembled isolation column and the collection vessel were subsequently centrifuged at 6.000x g for 30 seconds and the flow through was discarded as described above. This washing procedure (adding ethanol to the column and subsequent centrifugation) was repeated three times. After the last washing step the column was placed in an empty collection vessel and was centrifuged at 16.000x g for 2 minutes.

6. Elution:

The isolation column was placed in a new 1.5 ml reaction container and 50 ul of RNase free water was added to the middle of the filter without touching the filter. The column containing the RNase free water is incubated for 1 minute at room temperature and is subsequently centrifuged at 6.000x g for 1 minute. The isolation column is discarded and the 1 .5 ml reaction container containing 50 μΙ of TNA-solution is closed.

Stabilization of blood with PAXgene and RNA isolation from PAXgene stabilized blood

For the PAXgene samples, each sample was prepared from 2.5 ml blood, drawn with a sodium citrate containing blood collection device, and mixed with 7.5 ml of a stabilization buffer containing 3% (w/v) tetradecyltrimethylammonium oxalate and 200 mM tartaric acid, respectively, in a 12 ml polyethylene tube. The pH of the buffer was adjusted with sodium hydroxide to 3.3, 3.5 and 3.7, respectively. Samples were stored at room temperature for 25 hours and 72 hours, respectively. In order to isolate the cellular RNA, the tubes were centrifuged at 5000 x g for 10 minutes. The supernatant was discarded and the pellet was washed once with water. After additional centrifugation at 5000 x g for 10 minutes, the pellet was dissolved in 300 μΙ of a lysis buffer, i.e., buffer RLT (QIAGEN GmbH), diluted with 360 μΙ water and 40 μΙ proteinase K were added. After a proteinase digestion for 10 minutes at 55°C the sample was centrifuged at 20,000 x g for 3 minutes, the supernatant was transferred into a new tube and 350 μΙ of 98% ethanol were added. The sample was then applied to a silica membrane containing spin column via centrifugation at 8000 x g for 1 minute. The spin column was washed once with a GITC containing washing buffer-like buffer RW1 (QIAGEN GmbH) and two times with an ethanol wash containing buffer-like buffer RPE (QIAGEN GmbH). The RNA was then eluted from the silica membrane with 2 x 40 μΙ of RNase free water.

Quantitative PCR analysis

Isolated RNA was reverse transcribed using MMLV reverse transcriptase. The resulting cDNA was quantified using gene specific primers and SYBR green based qPCR master mix on a Roche LightCycler 480 instrument.

Example 1 : Analysis of RNA stability after TNA isolation from stabilized blood The experiment provided herein shows that stabilization of blood samples in ammonium formate prevents degradation or loss of RNA in the sample over longer periods than PAXgene.

Blood samples were stabilized in PAXgene or ammonium formate and were incubated for 0-14 days at 20°C. After incubation, RNA was purified from the samples and transcribed into cDNA and expression of reference genes was measured by quantitative PCR.

Blood samples that were stabilized in PAXgene were incubated for 0, 1 , 2 or 3 days at 18-25°C. Expression of a reference gene was measured at each time point and fold changes in expression as compared to day 0 were analyzed. Equal amounts of analyzed RNA were found at each time point demonstrating that PAXgene could stabilize RNA in the samples for up to 3 days without significant degradation or loss.

Blood samples that were stabilized in ammonium formate were incubated for 0, 2, 5, 6, 13 or 14 days at 20°C. Expression of reference genes ACTB, C6orf64, RPL13A and RPL32 were analyzed at each time point and average fold changes in expression as compared to day 0.

Equal amounts of each analyzed RNA were found at each time point demonstrating that ammonium formate could stabilize RNA in the samples for up to 14 days without significant degradation or loss.

Example 2: Analysis of RNA quality after stabilization with ammonium in a urine sample

The experiment provided herein shows that RNA integrity in a urine sample that has been stabilized with ammonium formate remains for over 72 hours at room temperature.

RNA from the bacteriophage MS2 has been added to urine samples, which have subsequently been stabilized with ammonium formate. After 48 and 72 hours RNA in the sample has been transcribed to cDNA and presence of MS2 RNA has been analyzed by qPCR. Results indicated that the amount of MS2 RNA in the samples was equal after 48 and 72 hours incubation at room temperature, indicating that RNA degradation was prevented by stabilization with ammonium formate.

Example 3: Analysis of reproducibility of RNA isolation efficiency

The experiment provided herein shows that sample stabilization in ammonium formate results in higher amounts of isolated RNA from a sample and enhanced reproducibility of experimental results.

Multiple blood samples from different patients were collected and stabilized in ammonium formate and PAXgene and stored at 20 °C for 3 days. Subsequently, RNA was isolated from the samples and was transcribed to cDNA and relative expression of the PGK gene was determined by quantitative PCR. The results showed that stabilization of blood samples in ammonium formate resulted in significantly higher amounts of isolated RNA as compared to stabilization in PAXgene.

Example 4: Side-by-side comparison of ammonium formate and PAXgene

This example refers to the advantages of the ammonium formate as a nucleic acid stabilizer as compared to PAXgene. The minimum volume of ammonium formate (10 M) needed for sample stabilization in a 10 ml sample collection tube is 0.5 ml as compared to 7.5 ml of PAXgene. Consequently, the maximum volume of sample that can be collected is 9.5 ml for ammonium formate as compared to 2.5 ml for PAXgene.

Ammonium formate can stabilize RNA at room temperature for 6 days, PAXgene can stabilize RNA for 3 days at room temperature. These advantages of ammonium formate over PAXgene are summarized in Table A.

Table A:

Example 5: Stabilization of nucleic acids by ammonium formate and salts of other ammonium cations

This example demonstrates the stabilization of nucleic acids by ammonium formate (AF) or a formate salt in combination with a salt compising a primary, secondary or tertiary ammonium cation (AC) in human blood samples.

Fresh blood from different donors is mixed with either AF (AF samples) or with a formate salt in combination with a salt comprising a primary, secondary or tertiary AC (AC samples), in particular a combination is employed using a formate salt together with Trimethylammonium chloride, Met- Lys formate, Pyridinium chloride, Pyridinium formate, methylammonium chloride, ethylammonium chloride, dimethylammonium dimethylcarbamate, Dimethylammonium chloride, Diethylammonium chloride, Met-Lys formate, Trimethylammonium chloride, Trimethylammonium acetate,

Triethylammonium formate, Triethylammonium chloride and Triethylammonium acetate are tested. As control, fresh blood is collected and stored in EDTA as anticoagulant without further stabilisation (N-samples). AF-, AC- and N-samples are stored at ambient room temperature for 14 days. Samples are tested at various test time points for nucleic acid stability and integrity. Nucleic acids are extracted by a spin column based purification method and analysed by agarose gel electrophoresis and/or determination of RNA Integrity Number and by measuring gene expression with qRT-PCR (e.g. FOS, IL1 β).

Example 6: Stabilization of nucleic acids by ammonium formate and salts of other carboxylate anions

This example demonstrates the stabilization of nucleic acids by ammonium formate (AF) and other carboxylate anions (CA) in human blood samples. Fresh blood from different donors is mixed with either AF (AF samples) or with various ammonium carboxylate salts (CA samples), in particular ammonium propionate, ammonium butyrate, ammonium isobutyrate, ammonium pentanoate, ammonium isovalerate and ammonium hexanoate are tested. As control, fresh blood is collected and stored in EDTA as anticoagulant without further stabilisation (N-samples). AF-, CA- and N-samples are stored at ambient room temperature for 14 days. Samples are tested at various test time points for nucleic acid stability and integrity. Nucleic acids are extracted by a spin column based purification method and analysed by agarose gel electrophoresis and/or determination of RNA Integrity Number and by measuring gene expression with qRT-PCR (e.g. FOS, IL1 β).

Claims

1. Method for stabilizing nucleic acids in a sample, comprising treating said sample with one or more salts comprising at least one salt according to Formula I:

wherein R is H or an alkyl group comprising 1-6 C atoms and X⁺ is a cation.

2. Method according to claim 1 , wherein X⁺ is an ammonium cation.

3. Method according to any one of the preceding claims, wherein the sample is treated with at least a formic acid salt (formate) and an ammonium salt.

4. Method according to any one of the preceding claims, wherein the sample is treated with ammonium formate.

5. Method according to any one of the preceding claims, wherein the sample comprises RNA and/or DNA.

6. Method according to any one of the preceding claims, wherein the sample is a biological sample obtained from, or comprising, one or more biological cells, organisms or viruses, a bodily fluid of a mammal, such as blood, urine, sputum, cerebrospinal fluid (CSF), a body swab, body smear, or a tissue sample.

7. Method according to any one of the preceding claims, wherein the sample is a blood sample or derived from a blood sample, such as whole blood, plasma or serum.

8. Method according to any one of the preceding claims, wherein the treatment of the

sample with one or more salts leads to a formate concentration of 0.01 M to 10 M, preferably 0.05 M to 8 M, more preferably 0.1 M to 7 M, or 0.25 M to 6 M in said sample, and preferably wherein the treatment of the sample with one or more salts leads to an ammonium concentration of 0.01 M to 10 M, preferably 0.05 M to 8 M, more preferably 0.1 M to 7 M, or 0.25 M to 6 M in said sample.

9. Method according to any one of the preceding claims, wherein the sample is treated with an ammonium formate solution, preferably at a concentration of 1 - 20 M.

10. Method according to any one of the preceding claims, wherein the sample is treated with ammonium formate in a solid form, for example in crystalline form.

1 1. Method for the detection and/or isolation of a nucleic acid from a sample, wherein said sample comprises a formate concentration of 0.01 M to 10 M and an ammonium concentration of 0.01 M to 10 M.

12. Kit for the detection, isolation and/or stabilization of nucleic acids from a biological

sample, comprising one or more salts, comprising at least one salt according to Formula I:

wherein R is H or an alkyl group comprising 1-6 C atoms and X⁺ is a cation, and additional means for nucleic acid isolation selected from the group consisting of cationic compounds, detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, such as guanidinium isothiocyanate, guanidinium hydrochloride, sodium dodecylsulfate, polyoxyethylene sorbitan monolaurate, RNAse inhibitor proteins, chloroform, isoamyl- alcohol, isopropanol, methanol, ethanol, glacial acetic acid, phenol, filter columns and combinations thereof.

13. Kit according to the preceding claim, comprising one or more salts, comprising at least one salt comprising a formate anion and at least one salt comprising an ammonium ion, preferably ammonium formate.

14. Collection container configured for the isolation of a blood sample, wherein said container comprises one or more salts, comprising at least one salt according to Formula I:

wherein R is H or an alkyl group comprising 1-6 C atoms and X⁺ is a cation, and wherein said container:

comprises a device for attachment of said container to a cannula or syringe, is a syringe suitable for blood isolation,

exhibits an internal pressure less than atmospheric pressure, such as is suitable for drawing a pre-determined volume of sample into said container, and/or

comprises additionally detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, such as guanidinium isothiocyanate, guanidinium hydrochloride, sodium dodecylsulfate, polyoxyethylene sorbitan monolaurate, RNAse inhibitor proteins, and mixtures thereof, and/or A filter system containing nitro-cellulose, silica matrix, ferromagnetic spheres, a cup retrieve spill over, trehalose, fructose, lactose, mannose, poly-ethylen-glycol, glycerol, EDTA, TRIS, limonene, xylene, benzoyl, phenol, mineral oil, anilin, pyrol, citrate, and mixtures thereof.

15. Collection container according to the preceding claim, comprising one or more salts, comprising at least one salt comprising a formate anion and at least one salt comprising an ammonium ion, preferably ammonium formate.