WO2008145705A1

WO2008145705A1 - Butenedioic acid or derivatives thereof for treating a biological sample

Info

Publication number: WO2008145705A1
Application number: PCT/EP2008/056639
Authority: WO
Inventors: Vera HOLLÄNDER
Original assignee: Qiagen Gmbh
Priority date: 2007-05-31
Filing date: 2008-05-29
Publication date: 2008-12-04
Also published as: DE102007025275A1; EP2164962A1

Abstract

The invention relates to a method for treating a biological sample, comprising the preparation of a biological sample, and bringing the biological sample into contact with butenedioic acid or with a butenedioic acid derivative. The invention also relates to treated biological samples obtained by said method, the use of butenedioic acid or derivatives thereof, a treated biological sample and a kit.

Description

BUTENIC ACID OR DERIVATIVES FOR TREATING A BIOLOGICAL SAMPLE

Field of the invention

The present invention relates to a method for treating a biological sample, the treated biological sample obtainable by this method, the use of butenedioic acid or its derivatives, a treated biological sample and a kit.

Technical background

It has long been known that the genetic origin and functional ac- tivity of a cell can be determined and studied by studies of its nucleic acids. Analyzes of nucleic acids and proteins provide a direct indication of the cause of cell activity. Thus, they are potentially superior to indirect, conventional methods such as the detection of metabolic products. Therefore, in the future with a still wide spread of nucleic acid and protein analysis is expected. For example, molecular biological analyzes have already been used in many areas, for example in medical and clinical diagnostics, in pharmacy, in the development and evaluation of pharmaceuticals, in food analysis and in the monitoring of food production, in the agricultural industry in the breeding of Crops and livestock, in forensics, in environmental analysis and in many other research areas.

By analyzing the RNA, especially the mRNA in cells, the activities of genes can be determined directly. The quantitative analysis of transcriptional For example, patterning (mRNA patterns) in cells by modern molecular biology techniques, such as real-time reverse transcriptase PCR ("real-time RT PCR") or gene expression chip analyzes, allow recognition of incorrectly expressed genes, thereby, for example, metabolic disease The analysis of DNA from cells by molecular biological methods such as PCR, RFLP, AFLP or sequencing allows, for example, the detection of genetic defects or the determination of the HLA type and other genetic The analysis of genomic DNA and RNA is also used for the direct detection of infectious agents, such as viruses, bacteria, etc.

However, the prerequisite for the analysis of biomolecules, in particular of nucleic acids and proteins, in biological samples is the immediate stabilization of the biological sample, since the status (gene expression profile or protein pattern) of the components of the fresh samples which are important for the molecular-biological examination already exists immediately after taking the sample from its natural environment can change rapidly. A longer storage of the samples in an untreated condition, eg. Due to unintentionally delayed transport to a laboratory can falsify a molecular biological analysis or make it completely impossible. Also other biomolecules such. B. metabolites and the morphology of a sample can be adversely affected by storage in the untreated state.

Especially the nucleic acid status of a biological sample changes the more, the more time passes between the taking of the sample and its analysis. Particularly fast is the degradation of ribonucleic acids (RNA) by ubiquitous RNases. Likewise, in addition to the degradation of nucleic acids, it also leads to the induction of, for example, stress genes and thus to Synthesis of new mRNA molecules that also strongly alter the transcript pattern of the sample. Therefore, it is necessary to perform an immediate stabilization of the sample to obtain the Genexpressionspro to be examined. The same applies if a frozen biological sample is thawed for the purpose of its examination. Again, after thawing, there is a rapid change in nucleic acid status, particularly by degradation of biomolecules.

Not only for the analysis of nucleic acids, but also for detailed investigations of the proteome of a biological sample, an immediate stabilization of the sample is necessary, since also the protein pattern is changed immediately after taking the sample. This is done on the one hand by degradation or new synthesis, on the other hand but particularly quickly by changes in protein modifications, such as. B. phosphorylation / dephosphorylation.

In particular, in order to completely preserve the expression pattern in the biological sample at the time of sampling, the stabilization must begin as soon as possible after the addition of the solution, without prior pretreatment of the sample. For the stabilization of nucleic acids in compact tissue samples results in comparison to other biological samples a particular difficulty. Tissues are complex and heterogeneous in composition, ingredients and construction. For the stabilization of nucleic acids in compact tissue samples, the effect of the stabilizing reagent must not only unfold on the surface of the cells or within a cell layer, but also act deep within the multilayered sample material. In addition, very different tissue and / or cell types often have to be addressed within one and the same biological sample, for example in the cell structure, the membrane structure, the compartmentalizations and the biomolecules, for example, in terms of proteins, carbohydrates and / or fat content, distinguish.

Over the years, a variety of different fixation or stabilization or stabilization reagents and methods have been developed to fix or stabilize a wide range of different biological samples.

Thus, as a method of stabilization, the freezing of biological samples has long been known. The sample is immediately after removal from their natural environment at -80 ⁰ C and lower, z. B in liquid nitrogen, deep-frozen. The thus treated sample can then be stored almost indefinitely without changes in the integrity at about -70 ⁰ C.

In histological analyzes, tissue structures as well as cellular and subcellular structures must be preserved in their morphological appearance during storage. In the case of tissue samples, the fixation by means of formalin and optionally the subsequent embedding of the fixed samples in paraffin has long been known.

Other known stabilization methods include the use of certain stabilizing reagents. These stabilizing reagents include, for example, cationic detergents (US 5,010,184, US 5,300,545, WO-A-02/00599 and WO-A-02/00600) or highly concentrated ammonium sulfate solutions (US 6,204,375).

The disadvantage of the known from the prior art stabilization method, however, is that either the stabilization of biomolecules such as nucleic acids in the biological samples only unsatisfactory, and in particular

- A - the yield of these biomolecules from the conventionally stabilized samples is often low and / or that the morphology of the samples is not sufficiently maintained.

In the case of stabilization by freezing, there is the additional disadvantage that this stabilization method requires very complicated, logistical conditions, since thawing of the samples during transport, storage or during a wide variety of application or use processes must be prevented. In addition to the additional costs for special sample containers and for the permanent cooling of the samples, the use of liquid nitrogen is not only very cumbersome, but can only be carried out under special precautionary measures. In addition, a subsequent analysis of the frozen sample material, in particular individual components of the sample, usually very difficult. To reduce the disadvantages of processing frozen samples, so-called transition solutions are known from the prior art. First, the frozen tissue is transferred into a pre-cooled to -70 ⁰ C to -80 ⁰ C solutions and then stored therein for a few hours (at least 16 hours) at about -20 ⁰ C. Subsequently, the sample impregnated with the transition solution can then be heated to working temperatures of -4 ° C. up to room temperature for only a short period of time, for example at most for dividing the sample, without the nucleic acid status of the sample changing. Such transition solutions, known for example from WO-A-2004/72270, consist primarily of monohydric alcohols.

The present invention has for its object to overcome the disadvantages resulting from the prior art. In particular, the present invention has the object, a method for the treatment of a biological sample, preferably for stabilizing a biological sample to specify, with the biomolecules, in particular nucleic acids, proteins and Metabo liten, can be better stabilized in the biological sample. The amount of isolatable biomolecules should be increased and / or the quality of the biomolecules should be increased in comparison to the conventional stabilization methods, which in the case of nucleic acids, for example by gel analysis or by the number of PCR cycles until a certain amount of nucleic acid is reached can be determined to be improved. The morphology of the biological sample should also be well preserved by the stabilization process.

In addition, the present invention has the object to provide a method for stabilizing a biological sample, with both frozen and fresh biological samples at moderate temperature conditions, for example, at room temperature, are stabilized without affecting the expression profile or the proteome of the biological sample can.

Furthermore, the method for the treatment, preferably for the stabilization of a biological sample should lead to a treated, preferably stabilized biological sample which can be analyzed not only at moderate temperatures, for example at room temperature, but optionally before or after such an analysis for as long as possible can be stored at such moderate temperature conditions.

In the case of biomolecules, the term "stabilization" should preferably be understood to mean the inhibition of the degradation, the modification, the induction or the change in the activity of biomolecules. logical analysis of the biological samples should be understood by the term "stabilization" preferably preventing a significant change in the morphology of the samples.

A contribution to the solution of the abovementioned objects is provided by a method for the treatment of a biological sample, comprising the method steps

i) providing a biological sample, and

ii) contacting the biological sample with butenedioic acid or with a derivative of butenedioic acid.

Butenedioic acid exists in two different steric conformations, namely trans-butenedioic acid, also called fumaric acid, and cis-butenedioic acid, also called maleic acid. The structural formulas of the two conformations are as follows.

(I) (H) trans-butenedioic acid (fumaric acid) cis-butenedioic acid (maleic acid)

Surprisingly, it was found that by contacting a biological sample with butenedioic acid or with their derivatives, in particular with

Fumaric acid, maleic acid and derivatives thereof, in particular maleimides,

Biomolecules such as nucleic acids in the biological sample are better stabilized let it go. The butenedioic acid or its derivatives can also be added as an additive to conventional stabilizing or fixing compositions. The compositions with this additive are distinguished by a better stabilizing power of biomolecules in a biological sample, in particular by an improved stabilization capacity for nucleic acids, compared to the compositions without the additive.

The derivatives of maleic acid preferably have the structure (III)

on, in the X

a) an oxygen atom, b) a group of structure (IV)

\ _{2 2} /

OR ^{2 2} RO

(IV) c) or a group NR ¹ ,

where R ¹

an optionally nitrogen or halogen-substituted alkyl radical having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms and more preferably 1 to 3 carbon atoms. lenstoffatomen, for example, with 1, 2, 3, 4, 5 or 6 carbon atoms,

an optionally nitrogen or halogen-substituted aryl radical having 6 to 12 carbon atoms, for example 6, 7, 8,

9, 10, 11 or 12 carbon atoms,

a COOR ³ radical in which R ^{3 is} a hydrogen atom or an alkyl radical having 1 to 6 carbon atoms, preferably having 1 to 4 carbon atoms and more preferably having 1 to 3 carbon atoms, for example 1, 2, 3, 4, 5 or 6 carbon atoms, is a hydrogen atom or a -NR ⁴ R ⁵ radical in which R ⁴ and R ^{5 is} a hydrogen atom, an optionally nitrogen or halogen-substituted alkyl radical having 1 to 6 carbon atoms, preferably having 1 to 4 carbon atoms and particularly preferably having 1 to 3 carbon atoms, for example having 1, 2, 3, 4, 5 or 6 carbon atoms or an optionally nitrogen or halogen-substituted aryl radical having 6 to 12 carbon atoms, for example 6, 7, 8, 9 , 10, 11 or 12 carbon atoms, and

where R ²

a hydrogen atom, an optionally nitrogen or halogen-substituted alkyl radical having 1 to 6 carbon atoms, preferably having 1 to 4 carbon atoms and more preferably having 1 to 3 carbon atoms aryl, for example, with 1, 2, 3, 4, 5 or 6 carbon atoms, or an optionally nitrogen or halogen-substituted aryl radical having 6 to 12 carbon atoms, for example with 6, 7, 8, 9, 10, 11 or 12 carbon atoms,

is.

Preferred compounds of structure (III) are, in particular, compounds selected from the following structures (V), (VI) and (VII):

(V) (VI) (VII)

These compounds are preferably maleic anhydrides (III), maleimides (IV) and esters of maleic acid (VII). In addition, fumaric acid (I), also falling within the first definition, is fumaric acid (I).

The biological sample provided in method step i) can be a frozen or non-frozen biological sample, it being possible to use as biological sample all biological samples known to the person skilled in the art. Preferred biological samples are selected from the Group comprising biomolecules, for example natural, preferably isolated, linear, branched or circular nucleic acids such as RNA, in particular mRNA, siRNA, miRNA, snRNA, tRNA, hnRNA or ribozymes, DNA and the like, synthetic or modified nucleic acids, for example oligonucleotides, in particular for PCR used primers, probes or standards, nucleic acids labeled with digoxigenin, biotin or fluorescent dyes or so-called PNAs, natural, preferably isolated proteins or oligopeptides, synthetic or modified proteins or oligopeptides, for example antibodies coupled with fluorescence markers or enzymes , Hormones, metabolites and metabolites, growth factors, lipids, oligosaccharides, polysaccharides, proteoglucans, body fluids such as blood, sperm, cerebrospinal fluid, saliva, sputum, crusta phlogistica or urine, fluids obtained when working up blood such as serum or plasma, leucocyte fractions or "buffy coat", leech saliva, feces, smears, punctates, dandruff, hair, cutaneous fragments, forensic samples, food or environmental samples containing free or bound biomolecules, especially free ones or bound nucleic acids, metabolites, whole organisms, preferably whole non-living organisms, tissues of multicellulars, preferably of insects and mammals, in particular of humans, for example in the form of tissue sections or fragments or organs, isolated cells, for example in the form of more adherent or suspended cell cultures, organelles, for example chloroplasts or mitochondria, vesicles, cell nuclei or chromosomes, plants, plant parts, plant tissue or plant cells, bacteria, viruses, viroids, prions, yeasts and fungi.

As a non-frozen biological sample, preferably a freshly prepared biological sample is used in method step i) of the method according to the invention, for example a fresh tissue sample or freshly isolated blood sample. cells from a living or dead organism or, in the case of synthetic biomolecules as a biological sample, freshly synthesized nucleic acids or proteins. According to the invention, a "fresh" biological sample is preferably understood to mean a sample which, before it is brought into contact with the butenedioic acid or its derivative in method step ii), not more than 96 hours, preferably not more than 48 hours, more preferably not more than 24 hours before, more preferably not more than 10 hours, yet more preferably not more than 60 minutes and most preferably not more than 10 minutes before or, in the case of a synthetic biomolecule, synthesized However, the term "fresh" biological sample also includes those samples which have been taken within the abovementioned periods but which have been pretreated prior to contact with the butenedioic acid or its derivative, for example with conventional fixatives, such as formalin, with dyes such as eosin, with antibodies and dergle cozy. The preparation of fresh cell or tissue samples can be carried out by any preparation method known to the skilled person for this purpose, in the case of a tissue sample, for example by means of a scalpel, for example during an operation or an autopsy, in the case of a blood cell sample by centrifuging freshly drawn blood and like. In the case of using a fresh biological sample, the butenedioic acid or its derivative, or a composition comprising this compound as an additive, serves primarily as a stabilizing reagent or as a stabilizing composition.

As a frozen biological sample, in method step i) of the method according to the invention preferably a biological sample is used which, after having been isolated for example in the manner described above, before contacting with the butenedioic acid or with its derivative in method step ii ) are initially at a temperature of 0 ⁰ C or less, preferential has been cooled to temperatures of -20 ⁰ C or less, and most preferably to temperatures of -70 ⁰ C or less, such as by contacting with liquid nitrogen. If a biological sample frozen in this way is used in the process according to the invention, the butenedioic acid or its derivative or a composition which comprises this compound as an additive serves primarily as a transition reagent or as a transition composition.

In method step i), the biological sample is contacted with butenedioic acid or with its derivative, preferably with a compound of structure III.

If the group -X- is an NR ¹ group, the radical R ^{1 of} this compound of structure III, in addition to a hydrogen atom, may be an optionally substituted nitrogen or halogen-substituted alkyl radical with 1 to 6 carbon atoms. The term "nitrogen-substituted alkyl radical" is intended according to the invention to include those alkyl radicals in which one or more hydrogen atoms by NH ₂ -ReSt, NHR radicals or NR ₂ radicals, in which R is preferably an alkyl radical having 1 to 6 According to the invention, the term "halogen-substituted alkyl radical" is intended to encompass those alkyl radicals in which one or more hydrogen atoms are replaced by a halogen atom, preferably by Fluorine, chlorine, bromine or iodine, more preferably substituted by fluorine or chlorine, is / are.

Particularly preferred alkyl radicals radicals R ¹ are selected from the group comprising

-CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₃ , -CH (CHs) ₂ , -CH ₂ Cl, -CHCl ₂ , -CCl ₃ ,

-CH ₂ CH ₂ Cl, -CHClCH ₃ , -CH ₂ NH ₂ and -CH ₂ CH ₂ NH ₂ ,

wherein -CH ₃ and -CH ₂ CH _{3 are} most preferred.

Furthermore, the radical R ^{1 may} be an optionally nitrogen or halogen-substituted aryl radical having 6 to 12 carbon atoms. Again, the phrase "nitrogen or halogen substituted aryl radical" has the meaning described above in connection with the alkyl radical.

Particularly preferred aryl radicals are selected from the group comprising

-C ₆ H ₅ (phenyl radical),

-C ₆ H ₄ Cl,

-CH ₂ -C ₆ H ₅ (benzyl radical),

-CH ₂ -C ₆ H ₄ Cl,

-Ci ₀ H ₇ (naphthyl radical) and

the phenyl radical being most preferred. Also, R ^{1 may} be a COOR ³ radical, where R ^{3 is} a hydrogen atom or an alkyl radical having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms and more preferably 1 to 3 carbon atoms, for example 1 , 2, 3, 4, 5 or 6 carbon atoms.

Particularly preferred COOR3 radicals are selected from the group comprising

-COOH, -COOCH ₃ , and -COOCH ₂ H ₃ ,

wherein the radical -COOH is most preferred.

Finally, the radical R ^{1 can} also be a -NR ⁴ R ⁵ radical in which R ⁴ and R ^{5 are} a hydrogen atom, an optionally nitrogen or halogen-substituted alkyl radical having 1 to 6 carbon atoms or an optionally nitrogen or Is halogen-substituted aryl radical having 6 to 12 carbon atoms, where the expression "nitrogen or carbon-substituted alkyl radical" or "nitrogen or halogen-substituted aryl radical" has the abovementioned meaning.

Particularly preferred -NR ⁴ R ⁵ radical radicals are selected from the group comprising

-NH ₂ ,

-NHCH ₃ , -N (CH ₃ ) ₂ , -NH (CH ₂ CH ₃ ), and -N (CH ₂ CH ₃ ) ₂ , wherein the radical -NH _{2 is} most preferred.

The radical R ² in the structure III as well as in the structures IV, V, VI and VII may, in addition to a hydrogen atom, be an optionally nitrogen or halogen-substituted alkyl radical having 1 to 6 carbon atoms, wherein the formulation "nitrogen or halogen-substituted alkyl radical "has the abovementioned meaning and preferred alkyl radicals R ^{2 are} those alkyl radicals which have already been mentioned as preferred alkyl radicals in connection with the radical R ^1. The radical R may also be mentioned ² in the structure III and in the structures IV, V, VI and VII to an optionally nitrogen or halogen-substituted aryl radical having 6 to 12 carbon atoms, for example with 6, 7, 8, 9, 10, 11 or 12 carbon atoms act In principle, the radicals R ² in the structures III to VII may be identical or different.

Most preferred compounds of structure III according to the invention are selected from the group comprising maleic anhydride, maleimide, methylmaleimide and ethylmaleimide, with methylmaleimide and ethylmaleimide being the most preferred derivatives of maleic acid.

According to a particular embodiment of the process according to the invention, the butenedioic acid or its derivative as such or as such, ie without further solid or liquid constituents, is brought into contact with the biological sample. This procedure can be particularly advantageous when a liquid biological sample is to be treated. In this case, the butenedioic acid or its derivative may be dissolved or dispersed in a suitable amount in the biological sample, preferably dissolved, the concentration of the butenedioic acid or the derivative of the butenedioic acid in the liquid biological sample after contacting with it to the saturation concentration the butenedioic acid or the derivative of the butenedioic acid in the respective composition, but preferably in a range from 0.1 mmol to 10 mol / l, more preferably from 10 mmol / 1 to 5 mol / l and most preferably from 100 mmol / 1 to 1 mol / l. In the case of a solid biological sample, the solid sample may also be loaded in the solid butenedioic acid or its derivative.

According to another particular embodiment of the process according to the invention, however, the butenedioic acid or its derivative is brought into contact not only alone but in the form of an additive contained in a composition with the biological sample.

In this connection, it is preferred that the composition comprises, in addition to the butenedioic acid or its derivative, at least one further component selected from a solvent, an additive or a mixture thereof.

The solvent which may optionally be included in the composition may be any solvent, including solvents usually contained in compositions for the treatment, in particular for the stabilization of biological samples. The solvent is preferably a solvent selected from the group comprising water, monohydric alcohols (monools), polyhydric alcohols, aldehydes, ketones, dimethyl sulfoxide, aromatic hydrocarbons, halogenated hydrocarbons, ethers, carboxylic acids, carboxylic acid amides, nitriles, nitroalkanes, Esters or mixtures of at least two of these solvents.

Particular preference is given among these solvents to solvents selected from the group comprising water, methanol, ethanol, 1-propanol, 2-propanol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol, 2,4-pentanediol, ethylene glycol, propylene glycol, Diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, 1,2,3-propanetriol, 1,2,6-hexanetriol, 3-methyl-1,3,5-pentanetriol, 1,2,4-butanetriol, acetic acid tonitrile, acetone, anisole, benzonitrile, benzyl alcohol, 1-methoxy-2-propanol, quinoline, cyclohexanone, diacetin, dichloromethane, chloroform, diethyl ether, dimethyl ether, toluene, dimethyl ketone, diethyl ketone, dimethyl adipate, dimethyl carbonate, dimethyl sulphate, Dioxane, dimethyl sulfoxide, methyl acetate, ethyl acetate, benzoic acid, methyl benzoate, ethyl benzoate, ethylbenzene, formamide, glycerol triacetic acid, ethyl acetoacetate, methyl acetoacetate, N, N-diethylacetamide, N-methyl-N-ethylacetamide, N, N-dimethylacetamide, N, N- Dimethylformamide, N-methyl-N-ethylformamide, N, N-diethylformamide, N, N-dimethylthioformamide, N, N-diethylthioformamide, N-methyl-N-ethylthioformamide, N, N-dimethylacetamide d, N-methyl-N-ethylacetamide, N, N-diethylacetamide, nitroethane, nitromethyltoluene, triethyl phosphate or mixtures of at least two of these solvents.

The additive, which may optionally be included in the composition, may be any additive normally included in compositions for the treatment, in particular for the stabilization of biological samples. Preferred additives are selected from the group comprising salts, such as, for example, ammonium sulfite, ammonium sulfate, cesium sulfate, further ammonium salts, further sulfate salts, lithium nitrate, sodium acetate, potassium acetate, sodium chloride, potassium chloride or calcium chloride, osmotically active substances, such as mannitol, detergents , Inhibitors, which inhibit the degradation of nucleic acids or proteins, such as the protease Inihibor PMSF or the commercially available products ANTI-RNase (Ambion, St. Austin, USA), RNAsecure ^® (Ambion) or DEPC, alkylating agents, acetylating agents , Halogenating agents, nucleotides, nucleotide analogues, amino acids, amino acid-analogous compounds, betaine, viscous sity regulators, dyes, especially dyes for the specific staining of certain cell structures, buffer compounds, for example MES, HEPES, MOPS or TRIS, preservatives, complexing agents, such as EDTA or EGTA, reducing agents, such as 2-mercaptoethanol, dithiothreitol (DTT), hydrogen sulfide, ascorbic acid, NADPH, tricarboxyethylphosphine (TCEP) and hexamethylphosphoric triamide (Me2N) 3P, oxidants such as 5,5'-dithio-bis (2-nitrobenzenoic acid) (DTNB), substances which improve the permeability of cells, for example DMSO or DOPE, chaotropic substances such as guanidinium isothiocyanate or guanidinium hydrochloride, fixatives, cross-linking agents such as formaldehyde or glutaric dialdehyde, acetic acid or trichloroacetic acid, sugars, drying agents such as silica gel, phenol and phenol derivatives and mixtures of at least two, at least three, at least four, at least five or at least at least six of these additives.

When the composition described above is a liquid composition, the butenedioic acid or its derivative may be present in this liquid composition in a concentration up to the saturation concentration in the particular composition, but preferably in a concentration in the range of from 0.01 mmol to 10 mol / l, more preferably from 1 mmol / 1 to 5 mol / l, and most preferably from 10 mmol / l to 4 mol / l.

Furthermore, in the case of using the butenedioic acid or a derivative of the butenedioic acid in the form of the above-described composition, it is preferred that the composition

0 to 99.99 wt.%, More preferably 25 to 99 wt.% And most preferably 50 to 90 wt.% Of the solvent, 0 to 99.99% by weight, more preferably 22 to 99% by weight and most preferably 40 to 80% by weight of the additive, and

in the case of a liquid composition, the butenedioic acid or its derivative in the concentration ranges described above, and in the case of a solid composition, the butenedioic acid or its derivative in an amount ranging from 0.001 to 99.9% by weight, more preferably in the range of 0.01 to 90% by weight, and most preferably in an amount in the range of 0.1 to 80% by weight,

wherein the total amount of solvent, additive and compound butenedioic acid or derivative of butenedioic acid is 100 wt .-%.

The preparation of the composition from the solvent, the additive and the butenedioic acid or the derivative of the butenedioic acid is preferably carried out by simply mixing the components. If one of the components has a melting point above room temperature, it may be preferable to heat this component to the melting point and then to mix it with the other components. However, it is also conceivable that if one of the components is solid in the preparation of the composition and another component in liquid form, the solid component in the liquid component to solve. Thus, for example, a solid compound of structure III can be dissolved in a liquid additive or solvent.

The contacting of the composition with the biological sample in process step ii) is preferably carried out in the case of a liquid composition by immersing the biological sample in the composition so that the entire sample is impregnated by the composition can. As a biological sample, a fluid or isolated cells or z. For example, when a granular sample is used, it is contacted by mixing the biological sample with the composition or suspending the biological sample in the composition. Alternatively, at the selected temperature, solid compositions may be dissolved directly in a liquid biological sample (eg, blood, plasma, urine, saliva). In the case of a solid biological sample and a solid composition, the solid sample may also be incorporated in the solid composition.

It is further erfmdungsgemäß preferred that the bringing into contact the biological sample with the butenedioic acid or its derivative and with the composition comprising the butenedioic acid or its derivative at a temperature in the range from -80 ⁰ C to +80 ⁰ C, preferably in a range from 0 ⁰ C to +80 ⁰ C, more preferably in a range of 2, 3, 4, 5, 6, 7 or 8 ° C to +80 ⁰ C and more preferably in a range from 18 ° C to +80 ⁰ C, for example, at a temperature of at least -20 ⁰ C, -19 ° C, -18 ° C, -17 ° C, -16 ° C, -15 ° C, -14 ° C, - 13 ° C, -12 ° C, -I TC, -10 ⁰ C, -9 ° C, -8 ° C, -TC, -6 ° C, -5 ° C, -4 ° C, -3 ° C , -2 ° C, -FC, 0 ⁰ C, FC, 2 ° C, 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ⁰ C , I FC, 12 ° C, 13 ° C, 14 ° C, 15 ° C, 16 ° C, 17 ° C, 18 ° C, 19 ° C, 20 ⁰ C, 2FC, 22 ° C, room temperature, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C, 30 ⁰ C, 3FC, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, 40 ⁰ C, 4FC, 42 ° C, 43 ° C, 4 4 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C, 50 ⁰ C, 5FC, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C or 60 ⁰ C.

In this case, the formulation means that "contacting the biological sample with the butenedioic acid or with its derivative or with the composition comprising the butenedioic acid or its derivative at a temperature in a range from -80 ⁰ C to + 80 ⁰ C" or at one of the other temperatures mentioned above, that after contacting the biological Sample with the butenedioic acid or with its derivative or the composition, the temperature of the mixture thus obtained is within the above-mentioned temperatures. Thus, it may be, for example, that as the biological material is a deep-frozen to temperatures of less than -20 ⁰ C sample, for example a stored in liquid nitrogen sample is used, in which case such an amount of maleic acid or derivative, or of composition with is used at such a temperature that after contacting the frozen biological sample with the butenedioic acid or with its derivative or with the composition, the temperature of the mixture (and thus also the temperature of the biological sample) in the above-mentioned temperature range.

Furthermore, according to a particular embodiment of the method according to the invention, it may also be preferred for the biological sample to be in contact with the butenedioic acid or its derivative or with the composition comprising the butenedioic acid or its derivative in process step ii), preferably under the above-mentioned temperature conditions, nor in a subsequent process step ii) process step iii) at a temperature in a range of -80 ⁰ C to +80 ⁰ C, preferably in a range of 0 ⁰ C to +80 ⁰ C, still more preferably in a range of 2, 3, 4, 5, 6, 7 or 8 ° C to +80 ⁰ C and more preferably in a range of 18 ° C to +80 ⁰ C, for example at a temperature of at least - 20 ⁰ C, -19 ° C, -18 ° C, -17 ° C, -16 ° C, -15 ° C, -14 ° C, -13 ° C, -12 ° C, -I TC, - 10 ⁰ C, - 9 ° C, -8 ° C, -TC, -6 ° C, -5 ° C, -4 ° C, -3 ° C, -2 ° C, -FC, 0 ⁰ C, FC , 2 ° C, 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ⁰ C, I FC, 12 ° C, 13 ° C, 14 ° C, 15 ° C, 16 ° C, 17 ° C, 18 ° C, 19 ° C, 20 ⁰ C, 2FC, 22 ° C, room temperature, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C, 30 ⁰ C, 3FC, 32 ° C, 33 ° C, 34 ° C , 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, 40 ⁰ C, 4FC, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C , 48 ° C, 49 ° C, 50 ⁰ C, 5FC, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C or 60 ⁰ C. is stored, wherein such storage may be for a period of at least one day, at least two days, at least three days, at least one week, at least two weeks, at least one month, at least three months, at least six months or at least 12 months ,

The method according to the present invention enables the storage of a treated biological sample at refrigerator temperatures, at room temperature or at even higher temperatures, without any noticeable degradation of biomolecules such as nucleic acids or proteins in the biological sample. This represents a significant advantage over conventional stabilization methods, since the process can be carried out without the use of liquid nitrogen or freezers and the stabilized sample can be stored without the use of liquid nitrogen or freezers. In particular, for the purpose of a long-term archival samples, of course, as usual, even at low temperatures, for example at -20 ⁰ C or -80 ⁰ C, are stored, but this is not mandatory.

After the treatment according to the invention and, if appropriate, before or even after a possible storage step iii), the treated biological sample can also be embedded in suitable embedding means, for example in paraffin or the like, in order then to be able to make tissue sections which are suitable for histological examinations more easily from the biological sample.

Furthermore, according to a particular embodiment of the method according to the invention, it may be preferable for the process step i) and ii) to contain one more process step (iv) analysis of biomolecules in or from the biological sample contacted with the butenedioic acid or its derivative (or composition containing the butenedioic acid or its derivative) and / or histological analysis of the butenedioic acid or its derivative; rivat (or with a composition containing the butenedioic acid or its derivative) brought biological sample, but particularly preferably the analysis of biomolecules in or with the butenedioic acid or with its derivative (or with a composition containing the butenedioic acid or their derivative) in contact with a biological sample,

if appropriate, this process step iv) can also be carried out before or after storage in accordance with method step iii) described above.

A histological examination is preferably understood to mean any examination method which is suitable for analyzing the morphological state of a tissue, a tissue section, a cell or subcellular structures, for example by means of microscopy and if appropriate using dyeing or labeling techniques known to the person skilled in the art.

Suitable biomolecules which can be analyzed are all biomolecules known to the person skilled in the art, in particular natural, modified or synthetic nucleic acids, natural, modified or synthetic proteins or oligopeptides, hormones, growth factors, metabolites, metabolites, lipids, oligosaccharides or proteoglucans. Suitable nucleic acids are all nucleic acids known to the skilled worker, in particular ribonucleic acids (RNA), for example mRNA, siRNA, miRNA, snRNA, t-RNA, hnRNA or ribozymes, or deoxyribonucleic acids (DNA). In principle It may be any type of polynucleotide that is an N-glycoside or C-glycoside of a purine or pyrimidine base. The nucleic acid may be single, double or multi-stranded, linear, branched or circular. It may correspond to a molecule occurring in a cell, such as genomic DNA or messenger RNA (mRNA), or generated in vitro, such as Complementary DNA (cDNA), counterstrand RNA (aRNA), or synthetic nucleic acids. The nucleic acid may consist of a few subunits, at least two subunits, preferably eight or more subunits, such as oligonucleotides, several hundred subunits to several thousand subunits, such as certain expression vectors, or significantly more subunits, such as genomic DNA. Preferably, the nucleic acid contains the coding information for a polypeptide in functional association with regulatory sequences that allow expression of the polypeptide in the cell into which the nucleic acid is introduced or naturally present. Thus, in a preferred embodiment, the nucleic acid is an expression vector. In another embodiment, it is a pDNA (plasmid DNA), siRNA, siRNA duplexes or siRNA hetero-duplexes, the term "siRNA" being understood to mean ribonucleic acids of about 22 nucleotides in length, which may be cleaved a double-stranded RNA (dsRNA) by the enzyme "Dicer" arise and in the enzyme complex "RISC" (RNA-induced silencing complex) are incorporated.

However, most preferred as biomolecules are proteins and nucleic acids, with nucleic acids being particularly preferred and RNAs being most preferred.

The phrase "analysis of biomolecules in or from the biological sample contacted with the butenedioic acid or its derivative or with a composition containing the butenedioic acid or its derivative" means that the analysis is carried out both in situ and ex situ So for example after isolation of the biomolecules from the biological sample can be done. If biomolecules from a biological sample are to be isolated for the purpose of analysis, it may be advantageous, in particular in the case of cells, tissues or other complex or compact samples, first to homogenize the samples, this homogenization being effected by mechanical means, for example by means of cannulas Mortars, rotor-stator homogenizers, a ball mill or the like, chemically by the use of suitable lysis buffers, which usually contain detergents and / or chaotropic substances and / or phenol, by enzymatic means, for example using proteases, or by a Combination of these measures can be carried out.

For histological analysis or for the analysis of biomolecules in or from the biological sample, all methods of analysis known to the person skilled in the art may be used, preferably methods selected from the group comprising light microscopy, electron microscopy, confocal laser scanning microscopy Laser micro-dissection, scanning electron microscopy, Western blotting, Southern blotting, Northern blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, affinity chromatography, mutation analysis, polyacrylamide gel electrophoresis (PAGE), in particular Two-dimensional PAGE, HPLC, Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) Analysis, SAGE Analysis (Serial Analysis of Gene Expression), Fast Protein Liquid Chroma- tography), mass spectrometry, for example MALDI-TOFF mass spectrometry or SELDI mass spectrometry, microarray analysis, LiquiChip analysis, enzyme activity analysis, HLA typing, sequencing, Whole Genome Amplification (WGA), Whole Transcriptome Amplification (WTA), RT-PCR , Real-time PCR or RT PCR, RNase protection analysis or primer extension analysis. According to a particular embodiment of the method according to the invention, method step iv) comprises an analysis of nucleic acids in or from the biological sample and / or an analysis of proteins in or from the biological sample.

A contribution to the solution of the abovementioned objects is also provided by the biological sample treated by the method according to the invention.

The use of butenedioic acid or a derivative of butenedioic acid, preferably of a compound of structure III, also makes a further contribution to achieving the abovementioned objects

R ² R ²

in

in which X and R ^{2 are} as defined above in connection with the inventive method, for the treatment of a biological sample, in particular for the stabilization of biomolecules, more preferably of nucleic acids or proteins, more preferably of nucleic acids and most preferably of RNAs, such as for example, mRNAs, in or from a biological sample and / or for the histological analysis of a biological sample, wherein as compounds of the structure III, as biomolecules and as a biological sample those compounds of the structure III, biomolecules and biological samples are preferred, already in the Connection with the erfmdungsannten method have been described as preferred embodiments. A contribution to the solution of the objects mentioned at the outset is also provided by a kit comprising

(a) maleic acid or a derivative of maleic acid, preferably the above-described compound of structure III, as well as

(b) reagents for analyzing biomolecules in or from a biological sample and / or analyzing the morphology of a biological sample.

The butenedioic acid or its derivative may be present in the kit according to the invention as a single component, but it is also conceivable that the kit is present as an additive in a composition as described in connection with the inventive method.

The reagents for the analysis of biomolecules in or from a biological sample or for the analysis of the morphology of a biological sample may in principle be all reagents known to those skilled in the art, for or during the morphological analysis of a biological sample or for or in the analysis of Biomolecules can be used in or out of a biological sample. These reagents include, in particular, dyes for staining cells or cell components, antibodies optionally labeled with fluorescent dyes or enzymes, an absorption matrix such as DEAE cellulose or a silica membrane, substrates for enzymes, agarose gels, polyacrylamide gels, solvents such as ethanol, chaotropic reagents or phenol, aqueous buffer solutions, RNase-free water, lysis reagents, alcoholic solutions and the like. Furthermore, a kit according to the invention as a component may, for example, contain at least one device (c), for example in the form of a sealable vessel, for collecting or taking up a solid or liquid biological sample. The device may optionally contain butenedioic acid or its derivatives or a composition comprising butenedioic acid or derivatives thereof prior to the collection or uptake of the biological sample. Device (c) may be any vessel known to those skilled in the art for collecting or receiving a solid or liquid biology sample. Preferred vessels are, for example, Falcon tubes, Eppendorf tubes, Greiner tubes, or one of those disclosed in US Pat. Nos. 6,602,718, US 2004/0043505 A1, US 2005/0160701 A1, US 2003/0086830 A1, US Pat.

US 2003/0087423 Al or WO 2005/014173 Al described vessels.

A erfϊndungsgemäßes kit can thereby

(a) butenedioic acid or a derivative of butenedioic acid, preferably the above-described compound of structure III, as well as

(B) a device for collecting or receiving a solid or liquid biological sample, wherein the device may optionally contain at least one of the compounds mentioned under (a) or a composition comprising at least one compound mentioned under (a) prior to collection or uptake of the biological sample ,

Alternatively, another kit of the invention may contain the components described above. The use of the kit for the analysis of biomolecules in or from a biological sample and / or for the histological analysis of a biological sample also contributes to the solution of the abovementioned objects.

A further contribution to the solution of the abovementioned objects is also provided by a method for the treatment of a disease, comprising the method steps:

(a) diagnosis of the disease by a diagnostic method comprising the analysis of a biological sample by methods described above comprising method step i), ii) and iv), optionally also iii), as well as

(b) therapeutic treatment of the diagnosed disease.

The invention will now be explained in more detail with reference to non-limiting figures and examples.

FIG. 1 shows the Western blot produced in Example 3.

FIG. 2 shows the agarose gel obtained in Example 6.

FIG. 3 shows the agarose gel obtained in Example 8.

Examples

1. Stabilization of RNA in biological samples in the presence of N-

ethylmaleimide

Renal tissue of the rat was immediately after organ removal with 500μl of a composition consisting of DMSO and 50 mM N-ethylmaleimide or 30% phenol in DMSO and 50 mM N-ethylmaleimide and stored at different temperatures (see Table 1). N-ethylmaleimide has the following structural formula (VIII):

CH ₂ CH ₃

(VIII)

Following storage, the RNA is isolated from the stored samples.

For this purpose, the tissue is removed after storage from the solutions and each 5 mg tissue 500 ul of a commercial guanidinium isothiocyanate buffer such. B. RLT buffer the company QIAGEN admit. The sample is removed by means of a ball mill, such. B. Tissue Lyzer of QIAGEN, homogenized over a period of 2 x 5 min at 25 Hz with a 5 mm steel ball, the guanidinium isothiocyanate buffer on known from the prior art, the cells lysiert and denature the liberated proteins , Subsequently, the lysates are centrifuged at 14000 rpm for 3 min. From the supernatant, 500 .mu.l, the 5 mg tissue, decreased. To these samples, 1 volume (500 μl) of 70% ethanol is added and mixed by repeated pipetting up or down or by vortexing for a period of about 5 seconds. The lysate is then in a commercially available silica membrane contained 96well plate such. B. Rneasy 96-plate from QIAGEN, and passed through the membrane by centrifugation (4 min at 6000 rpm). The RNA remains bound to the membrane and is subsequently washed with a first commercially available guanidinium isothiocyanate-containing washing buffer, for example with the buffer RWI from QIAGEN. Subsequently, for the enzymatic removal of any bound total DNA, DNA is placed in a suitable buffer on the column and incubated for 15 min at room temperature for the purpose of degrading the bound DNA. Following is again with a first commercially available guanidinium isothiocyanate-containing wash buffer, for example with the buffer RWI from QIAGEN, and then with a second tris-containing or tris and alcohol-containing wash buffer, z. B. Buffer RPE from QIAGEN, washed. In each case, the washing buffers are passed through the membrane by centrifugation (4 min at 6000 rpm). The washing with the second Tris-containing or Tris- and alcohol-containing wash buffer is repeated, at the same time the membrane by the centrifugation (10 min 6000 rpm) is dried. For elution, 50 μl of RNase-free water are pipetted onto the membrane to detach the purified RNA from the membrane. After incubation for 1 min at a temperature in the range 10-30 ⁰ C, the eluate is collected by centrifugation (1 min at 10,000 × g) passed through the membrane and the elution step is repeated once more to complete the elution.

The amount of isolated total RNA is determined after dilution in water by photometric measurement of light absorption at a wavelength of 260 nm. The quality of the RNA thus obtained is determined by the photometric Determination of the ratio of light absorption at 260 nm to that determined at 280 nm. The results of the insulations are shown in Table 1.

Table 1

As can be seen from Table 1, by adding N-ethylmaleimide as an additive in stabilizing compositions, biological samples can be stored for long periods of time at moderate temperatures and still sufficient quantities of good quality RNA can be isolated from the samples.

2. Stabilization of DNA in biological samples in the presence of N-ethylmaleimide

Renal tissue of the rat was immediately after organ removal with 500 ul DMSO + 50 mM N-ethylmaleimide or 30% phenol in DMSO + 50 mM N-ethylmaleimide and stored at different temperatures (see Table 2). Following storage, the DNA is isolated from the stored samples.

For DNA isolation, the tissue is removed after storage from the solutions and each 10 mg of tissue in 180 ul of the buffer ALT manufacturer QIAGEN add. The sample is removed by means of a ball mill, such. B. Tissue Lyzer QIAGEN, homogenized over a period of 30 s at 25 Hz with a 5 mm steel ball and then centrifuged for 15 s at 14000 xg. After addition of 120 .mu.l of the protease K solution (manufacturer QIAGEN), the lysates are incubated for 2 hours at 55.degree. C. with shaking. After incubation add 4 μl RNAse A (100 mg / ml), mix and incubate the mixture for 2 min at room temperature. After incubation, 300 .mu.l of a commercially available guanidinium hydrochloride-containing lysis buffer, such as the buffer AL of the manufacturer QIAGEN, is added and the samples are mixed by vortexing. There is an incubation at 70 ⁰ C for 10 min. After mixing with 300 .mu.l of 100% ethanol, the samples are applied to a 96-well plate (DNeasy 96-plate from QIAGEN) containing silica, and the lysate is passed through the membrane by centrifugation for 10 min at 6000 rpm. The DNA remains bound to the membrane and is first washed with a first commercially available guanidinium hydrochloride-containing wash buffer, for example with the buffer AWI from QIAGEN, and then with a second alcohol-containing wash buffer, z. B. buffer AW2 from QIAGEN, washed. The wash buffers are each by centrifugation (5 min at 6000 rpm) through the Passed membrane. Subsequently, the plate is incubated for 10 min at 70 ⁰ C. The elution of the DNA is carried out by application of 200 .mu.l of the pre-warmed to 70 ⁰ C elution buffer AE (QIAGEN). After incubation for 1 minute, the elution buffer is passed through the membrane by centrifugation (5 min at 6000 rpm) and the elution is repeated.

The amount of isolated total DNA is determined after dilution in water by photometric measurement of light absorption at a wavelength of 260 nm. The quality of the DNA thus obtained is determined by the photometric determination of the ratio of light absorption at 260 nm to that at 280 nm. The results of the insulations are shown in Table 2.

Table 2

As can be seen from Table 2, by adding N-ethylmaleimide as an additive in stabilizing compositions, biological samples can be stored for long periods of time at moderate temperatures and still sufficient quantities of DNA can be isolated from the samples in good quality.

3. Stabilization of proteins in biological samples in the presence of N-ethylmaleimide Rat liver tissue was promptly harvested with 1 ml each of 30% phenol in DMSO + 50 mM N-ethylmaleimide (Sample 1), DMSO + 50 mM N-ethylmaleimide (Sample 2) and 100% DMSO (as a negative control, Sample 3) immediately after organ removal Stored for 3 days at room temperature. Following storage, a protein extract is prepared from the stored samples.

To prepare the protein extract, the tissue is removed from the solutions after storage, and 10 .mu.l tissue each of 400 .mu.l of a conventional extraction buffer, here in a composition of 8 M urea, 100 mM sodium dihydrogen phosphate and 10 mM Tris, pH 8.0, add and sample with the aid of a ball mill, z. B. the Tissue Lyzer QIAGEN, homogenized. The resulting lysate is centrifuged for 15 s at the highest possible speed (eg about 20,000 x g) in order to pellet undissolved constituents. The proteinaceous supernatant is removed and the protein concentration is determined by means of a Bradford test. 1.5 μg protein in each case is separated on an SDS-polyacrylamide gel according to the customary procedure and blotted to a nitrocellulose membrane by means of a mid-blotting apparatus according to the manufacturer's instructions. The membrane is saturated with milk powder according to the state of the art and hybridized with an ERK2-specific antibody and a tubulin-specific antibody according to the manufacturer's instructions, and an immunodetection is carried out. The results are shown in FIG.

The detection of specific proteins proves that the proteins are stabilized by the N-ethylmaleimide-containing composition at room temperature in tissues, while without the addition of this additive (lane 3) no protein is detectable. 4. Transition of frozen biological samples in the presence of N-ethylmaleimide

Liver tissue from rat, which remain frozen after removal in liquid nitrogen and stored at -70 ⁰ C, is used for this experiment. For each transition experiment, 20 to 50 mg of tissue are weighed and frozen with various non-cooled (room temperature) Transistionssreagenzien (DMSO or mixtures of DMSO and phenol) and stored for 3 days at room temperature. For the transition, on the one hand 1 ml of the solution are used directly and on the other hand 950 μl of the solution mixed with 50 μl of 1 M N-ethylmaleimide to a final concentration of 50 mM NEM (solutions used see Table 3). Following the transition, the RNA is isolated from the stored samples.

For this purpose, the tissue is removed after storage from the treatment solutions and each 10 mg tissue 350 ul of a commercial guanidinium isothiocyanate buffer such. B. RLT buffer the company QIAGEN admit. The sample is removed by means of a ball mill, such. B. MM300 QIAGEN, homogenized over a period of 2 x 2 min at 20 Hz with a 5 mm steel ball, wherein the guanidinium isothiocyanate buffer on known from the prior art, the cells lysiert and denature the released proteins. Subsequently, the lysates are centrifuged at 14000 rpm for 3 min. From the supernatant, two portions of 350 μl each, corresponding to 10 mg of tissue, are removed. 1 volume (350 μl) of 70% ethanol is added to these samples and mixed by repeated pipetting up or down or by vortexing over a period of about 5 seconds. The lysate is then incorporated into a commercially available silica membrane spin column, such as. B. RNeasy columns from QIAGEN, applied and passed through the membrane by centrifugation (1 min at 10,000 xg). The RNA remains bound to the membrane and is then treated with a first commercially available guanidinium isothiocyanate-containing wash buffer, for example with the buffer RWI from QIAGEN, and then with a second Tris-containing or Tris and alcohol-containing wash buffer, z. B. Buffer RPE from QIAGEN, washed. In each case, the washing buffers are passed through the membrane by centrifugation (1 min at 10,000 × g). The washing with the second Tris-containing or alcoholic washing buffer is repeated with a smaller volume, while the membrane is simultaneously dried by centrifugation (2 min at 20,000 × g). For elution, 40 μl of RNase-free water are pipetted onto the membrane to detach the purified RNA from the membrane. After incubation for 1 min. at a temperature in the range of 10 - 30 ⁰ C, the eluate is passed through the membrane by centrifugation (1 min at 10,000 xg) and the elution step is repeated once more for the purpose of complete elution.

Subsequently, the amount and quality of isolated total RNA are determined, as described in Example 1. The results are also shown in Table 3.

Table 3

Table 3 shows that N-ethylmaleimide also has a positive influence on the stabilization of RNA in biological samples in transition solutions.

5. Transition of frozen biological samples in the presence of N-ethylmaleimide

Liver tissue from rat, which was frozen after removal in liquid nitrogen and stored at -70 ⁰ C, is used for this experiment. For each transition experiment, 20 to 50 mg of tissue are weighed and frozen with various non-cooled (room temperature) transition reagents and stored for 3 days at 4 ° C. On the one hand, 1 ml of the solution is used directly for the transition and, on the other hand, 950 μl of the solution are mixed with 100 μl of IM N-ethylmaleimide (for solutions, see Table 4). Following the transition, the RNA is isolated from the stored samples and the amount and quality of the RNA determined as described in Example 4. The results are also shown in Table 4.

Table 4

Table 4 also shows that N-ethylmaleimide in transition solutions has a positive influence on the stabilization of RNA in biological samples.

6. Transition of biological samples in the presence of N-ethylmaleimide

Kidney tissue from rat, which was frozen after removal in liquid nitrogen and stored at -70 ⁰ C, is used for this experiment. For each transition experiment, 10 to 30 mg of tissue are weighed and freeze-dried with various non-cooled (room temperature) transient reagents and stored for 5 days at room temperature. For the transition, firstly 1 ml of the solution is mixed with 50 μl of 1 M N-ethylmaleimide (solutions used, see Table 5). Following the transition, the RNA is isolated from the stored samples and the amount and quality of the RNA determined as described in Example 4. The results are also shown in Table 5.

Table 5

Table 5 also shows that the addition of N-ethylmaleimide in the transition solutions described here improves the stabilization of RNA in biological samples.

The isolated RNA is analyzed on agarose gels stained with ethidium bromide. For example, 1.0% formaldehyde-agarose MOPS gel is prepared for this purpose. In each case 5 .mu.l of the eluate are used. The result is shown in FIG. From this Figure 2 it can be seen that N-ethylmaleimide also has a positive influence on the quality of the RNA.

7. Transition of biological samples in the presence of various maleimide derivatives

Liver tissue from rat, which remain frozen after removal in liquid nitrogen and stored at -70 ⁰ C, is used for this experiment. For each

Transition experiment, 10 to 30 mg of tissue are weighed and frozen with various, not pre-cooled (room temperature) Transitionsreagenzien added and stored for 3 days at room temperature. For stabilization, 0.5 ml of 100% DMSO is used as a control. On the other hand, 0.5 ml each of a solution of DMSO and the various additives dissolved in A. dest are prepared (for solutions, see Table 6). Following storage, the RNA is isolated from the stored samples and the amount and quality of the isolated RNA determined as described in Example 4. The results are also shown in Table 6.

Table 6

Table 6 shows that, in addition to N-ethylmaleimide, N-methylmaleimide, maleic acid and fumaric acid in transition solutions also have a positive influence on the stabilization of RNA in biological samples.

N-methylmaleimide has the following structural formula (IX):

(IX) 8. Storage of biological samples outside the treatment compositions

Rat liver tissue that was frozen after removal in liquid nitrogen and stored at -70 ⁰ C, is used for this experiment. As a transition composition, a solution of 30% phenol dissolved in DMSO is prepared and mixed 1,350 ul of this solution with 150 ul IM NEM, so that the final concentration of NEM is 100 mM. For the transition experiment, approx. 150 mg of tissue are weighed and frozen with the solution pre-cooled in the refrigerator to 2 to 8 ° C. The sample is stored overnight at 2 to 8 ° C in the refrigerator.

After the transition, the samples are taken from the transition composition, dissected and stored in parts (about 10-30 mg) dry at room temperature for 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours 30 minutes and 4 hours 30 minutes. Subsequently, the RNA, as described in Example 4, isolated and, as described in Example 6, applied to an agarose gel. The result is shown in FIG.

It can be seen from FIG. 3 that the samples stabilized according to the invention can also be stored for a long time outside the treatment compositions without the quality of the RNA isolated from these samples being appreciably impaired.

9. Transition of biological samples in the presence of various maleimide derivatives

Rat liver tissue that was frozen after removal in liquid nitrogen and stored at -70 ⁰ C, is used for this experiment. For the Transition experiments are weighed 15 to 50 mg of tissue and frozen with IM N-ethylmaleimide or with 0.2 M N-methylmaleimide, each dissolved in water, and stored at 4 ° C or room temperature. The transition solutions are not precooled before use, ie they are used at room temperature. Following storage, the RNA is isolated from the stored samples and the amount and quality of the isolated RNA determined as described in Example 4. The results are shown in Table 7.

Table 7:

It can be seen from Table 7 that N-ethylmaleimide and N-methylmaleimide, even in water which itself has no stabilizing properties, as transition solutions enable the stabilization of RNA at moderate temperatures in biological samples.

10. Stabilization of biological samples in the presence of fumaric acids

Rat liver tissue was immediately after organ removal in mixtures of polyols and fumaric acid (see Table 8) and stored for 3 days at room temperature. The fumaric acid was dissolved 10% in ethanol and washed with the in the polyol compositions listed in Table 8. Following storage, the RNA is isolated from the stored samples and the amount and quality of the isolated RNA determined as described in Example 4. The results are shown in Table 8.

Table 8:

The examples described above show that fumaric acid, maleic acid, maleic acid derivatives and maleimides such as N-ethylmaleimide or N-methylmaleimide in various compositions have a positive influence on the stabilization of biomolecules, in particular of nucleic acids or proteins, both in the treatment of fresh, not -forforced biological samples as well as in the treatment of frozen biological samples.

Claims

1. A method for treating a biological sample, comprising the method steps

i) providing a biological sample, and

2. The method of claim 1, wherein the derivative of the butenedioic acid is a maleic acid derivative having the structure (III)

has, in the

in the X

a) an oxygen atom, b) a group of structure (IV)

\ _{2 2} /

OR ^{2 2} RO

(IV) c) or a group NR ¹ ,

where R

an optionally nitrogen or halogen substituted alkyl radical

Radical having 1 to 6 carbon atoms, preferably having 1 to 4 carbon atoms and more preferably having 1 to 3 carbon atoms, for example having 1, 2, 3, 4, 5 or 6 carbon atoms,

an optionally nitrogen or halogen-substituted aryl radical having 6 to 12 carbon atoms, for example having 6, 7, 8, 9, 10, 11 or 12 carbon atoms,

a COOR ³ radical in which R ^{3 is} a hydrogen atom or an alkyl radical having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms and more preferably 1 to 3 carbon atoms, for example 1, 2, 3, 4 , 5 or 6 carbon atoms, - is a hydrogen atom or an -NR ⁴ R ⁵ radical in which R ⁴ and R ^{5 is} a hydrogen atom, an optionally nitrogen or halogen-substituted alkyl radical having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms and particularly preferably with 1 to 3 carbon atoms, for example with 1, 2, 3, 4, 5 or 6 carbon atoms or an optionally nitrogen or halogen-substituted aryl radical having 6 to 12 carbon atoms, for example 6, 7, 8 , 9, 10, 11 or 12 carbon atoms, and where R ²

a hydrogen atom, an optionally nitrogen or halogen-substituted alkyl radical having 1 to 6 carbon atoms, preferably 1 to 4

Carbon atoms and especially preferably having 1 to 3 carbon atoms, for example having 1, 2, 3, 4, 5 or 6 carbon atoms, or an optionally nitrogen or halogen-substituted aryl radical having 6 to 12 carbon atoms, for example 6, 7, 8,

9, 10, 11 or 12 carbon atoms,

is.

The method of claim 1 or 2, wherein the biological sample is a frozen or a non-frozen biological sample.

4. The method according to any one of the preceding claims, wherein the butenedioic acid or its derivative in the form of a composition comprising the butenedioic acid or its derivative and at least one further component selected from a solvent, an additive or a mixture thereof.

5. The method of claim 4, wherein the solvent is a solvent selected from the group consisting of water, monohydric or polyhydric alcohols, aldehydes, ketones, dimethyl sulfoxide, aromatic hydrocarbons, halogenated hydrocarbons, ethers, carboxylic acids, carboxylic acid amides, nitriles, nitroalkanes , Esters or mixtures of at least two of these solvents.

A process according to any one of the preceding claims, wherein the composition is a liquid composition comprising the butenedioic acid or its derivative in a concentration ranging from 0.01 mmol / l to 10 mol / l up to the saturation concentration.

7. The method according to any one of claims 4 to 6, wherein the additive is selected from the group comprising salts, detergents, osmotic active substances, inhibitors which inhibit the degradation of nucleic acids or proteins, viscosity regulators, dyes, buffer compounds, preservatives, complexing agents , Reducing agents, substances which improve the permeability of cells, chaotropic substances, sugars, phenol or phenol derivatives, drying agents, fixatives and mixtures of at least two of these additives.

8. The method according to any one of the preceding claims, wherein it is the derivative of the butenedioic acid to fumaric acid, maleic anhydride, maleimide, N-ethylmaleimide, N-methylmaleimide or mixtures of at least two of these compounds.

9. The method according to any one of the preceding claims, wherein the bringing into contact of the biological sample with the butenedioic acid or its derivative at a temperature in a range of -80 ⁰ C to +80 ⁰ C.

10. The method according to any one of the preceding claims, wherein the method in addition to the process steps i and ii) nor the process step iii) storage of the brought into contact with the butenedioic acid or derivative thereof with the biological sample at a temperature in the range from -80 ⁰ C to +80 ⁰ C

and / or the method step:

vi) Analysis of biomolecules in or from the butenedioic acid or biological sample brought into contact with its derivative and / or histological analysis of the biological sample contacted with the butenedioic acid or with its derivative

includes.

11. A treated biological sample obtainable by the method according to any one of claims 1 to 10.

12. Use of butenedioic acid or a derivative of butenedioic acid, preferably a compound of structure (III)

in the X

d) an oxygen atom, e) a group of structure (IV) \ _{2 2} /

OR ^{2 2} RO

(IV) f) or a group NR ¹ ,

where R

an optionally nitrogen or halogen-substituted alkyl radical having 1 to 6 carbon atoms, preferably having 1 to 4 carbon atoms and particularly preferably having 1 to 3 carbon atoms, for example having 1, 2, 3, 4, 5 or 6 carbon atoms,

a COOR ³ radical in which R ^{3 is} a hydrogen atom or an alkyl radical having 1 to 6 carbon atoms, preferably having 1 to 4 carbon atoms and more preferably having 1 to 3 carbon atoms, for example 1, 2, 3, 4, 5 or 6 carbon atoms - is a hydrogen atom or a -NR ⁴ R ⁵ radical in which R ⁴ and R ^{5 is} a hydrogen atom, an optionally nitrogen or halogen-substituted alkyl radical having 1 to 6 carbon atoms, preferably having 1 to 4 carbon atoms and especially preferably having 1 to 3 carbon atoms, for example having 1, 2, 3, 4, 5 or 6 carbon atoms or an optionally nitrogen or halogen-substituted aryl radical having 6 to 12 carbon atoms, for example 6, 7, 8, 9, 10, 11 or 12 carbon atoms, and where R ²

a hydrogen atom, an optionally nitrogen or halogen-substituted alkyl radical having 1 to 6 carbon atoms, preferably having 1 to 4 carbon atoms and particularly preferably having 1 to 3 carbon atoms, for example having 1, 2, 3, 4, 5 or 6 carbon atoms, or optionally substituted by nitrogen or halogen aryl radical having 6 to 12 carbon atoms, for example, with 6, 7, 8, 9, 10, 11 or 12 carbon atoms,

is

for the treatment of a biological sample, in particular for the stabilization of biomolecules in a biological sample.

13. A kit comprising

(a) a composition comprising butenedioic acid or a derivative of butenedioic acid as defined in any one of claims 2, 8 and 12, and optionally

(b) at least one reagent for analyzing biomolecules in or from a biological sample and / or for analyzing the morphology of a biological sample, and / or

(c) at least one optionally closable device for collecting or receiving a biological sample, where Composition (a) may be included prior to collection or uptake of the biological sample.