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WO2008145705A1 - Acide butènedioïque ou ses dérivés pour le traitement d'un échantillon biologique - Google Patents

Acide butènedioïque ou ses dérivés pour le traitement d'un échantillon biologique Download PDF

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Publication number
WO2008145705A1
WO2008145705A1 PCT/EP2008/056639 EP2008056639W WO2008145705A1 WO 2008145705 A1 WO2008145705 A1 WO 2008145705A1 EP 2008056639 W EP2008056639 W EP 2008056639W WO 2008145705 A1 WO2008145705 A1 WO 2008145705A1
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WIPO (PCT)
Prior art keywords
carbon atoms
biological sample
derivative
butenedioic acid
halogen
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Application number
PCT/EP2008/056639
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German (de)
English (en)
Inventor
Vera HOLLÄNDER
Original Assignee
Qiagen Gmbh
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Application filed by Qiagen Gmbh filed Critical Qiagen Gmbh
Priority to EP08760230A priority Critical patent/EP2164962A1/fr
Publication of WO2008145705A1 publication Critical patent/WO2008145705A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • 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.
  • nucleic acid and protein analysis 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.
  • RNA patterns 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
  • mRNA patterns patterning in cells by modern molecular biology techniques, such as real-time reverse transcriptase PCR ("real-time RT PCR") or gene expression chip analyzes
  • real-time RT PCR” real-time reverse transcriptase PCR
  • 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
  • genomic DNA and RNA is also used for the direct detection of infectious agents, such as viruses, bacteria, etc.
  • 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.
  • RNA ribonucleic acids
  • the stabilization 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • tissue structures as well as cellular and subcellular structures must be preserved in their morphological appearance during storage.
  • fixation by means of formalin and optionally the subsequent embedding of the fixed samples in paraffin has long been known.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Butenedioic acid exists in two different steric conformations, namely trans-butenedioic acid, also called fumaric acid, and cis-butenedioic acid, also called maleic acid.
  • trans-butenedioic acid also called fumaric acid
  • cis-butenedioic acid also called maleic acid.
  • the structural formulas of the two conformations are as follows.
  • 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)
  • 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,
  • 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
  • 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,
  • Preferred compounds of structure (III) are, in particular, compounds selected from the following structures (V), (VI) and (VII):
  • fumaric acid (I) 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, polysacchari
  • 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.
  • 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.
  • 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
  • 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.
  • a tissue sample for example by means of a scalpel, for example during an operation or an autopsy
  • a blood cell sample by centrifuging freshly drawn blood and like.
  • 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.
  • 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.
  • the biological sample is contacted with butenedioic acid or with its derivative, preferably with a compound of structure III.
  • 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.
  • 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
  • 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
  • the radical R 1 may be an optionally nitrogen or halogen-substituted aryl radical having 6 to 12 carbon atoms.
  • nitrogen or halogen substituted aryl radical has the meaning described above in connection with the alkyl radical.
  • aryl radicals are selected from the group comprising
  • 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.
  • COOR3 radicals are selected from the group comprising
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • the solid sample may also be loaded in the solid butenedioic acid or its derivative.
  • 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.
  • 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.
  • 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
  • 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.
  • salts such as, for example, ammonium sulfite, ammonium sulfate, cesium sulfate, further ammonium salts, further sulfate salts, lithium nitrate,
  • RNAsecure ® (Ambion) or DEPC
  • alkylating agents 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
  • 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.
  • the composition 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
  • 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,
  • 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.
  • a biological sample a fluid or isolated cells or z.
  • solid compositions may be dissolved directly in a liquid biological sample (eg, blood, plasma, urine, saliva).
  • a solid biological sample and a solid composition the solid sample may also be incorporated in the solid composition.
  • 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.
  • the biological material is a deep-frozen to temperatures of less than -20 0 C sample
  • 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.
  • the biological sample 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the process step i) and ii) 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,
  • 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).
  • RNA ribonucleic acids
  • 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.
  • mRNA messenger RNA
  • cDNA Complementary DNA
  • aRNA counterstrand RNA
  • 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.
  • 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.
  • the nucleic acid is an expression vector.
  • siRNA in another embodiment, it is a pDNA (plasmid DNA), siRNA, siRNA duplexes or siRNA hetero-duplexes
  • 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.
  • biomolecules are proteins and nucleic acids, with nucleic acids being particularly preferred and RNAs being most preferred.
  • 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.
  • 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.
  • mechanical means for example by means of cannulas Mortars, rotor-stator homogenizers, a ball mill or the like
  • suitable lysis buffers which usually contain detergents and / or chaotropic substances and / or phenol
  • enzymatic means for example using proteases, or by a Combination of these measures can be carried out.
  • 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
  • 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
  • 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.
  • kits 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.
  • a erf ⁇ ndungshieles kit can thereby
  • 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.
  • 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
  • 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.
  • N-ethylmaleimide has the following structural formula (VIII):
  • RNA is isolated from the stored samples.
  • 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.
  • RNA 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.
  • 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.
  • a suitable buffer on the column 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.
  • 50 ⁇ l of RNase-free water are pipetted onto the membrane to detach the purified RNA from the membrane.
  • 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.
  • 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.
  • 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.
  • 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.
  • 120 .mu.l of the protease K solution manufactured by manufactureurer QIAGEN
  • the lysates are incubated for 2 hours at 55.degree. C. with shaking.
  • After incubation add 4 ⁇ l RNAse A (100 mg / ml), mix and incubate the mixture for 2 min at room temperature.
  • a commercially available guanidinium hydrochloride-containing lysis buffer such as the buffer AL of the manufacturer QIAGEN
  • the samples are mixed by vortexing. There is an incubation at 70 0 C for 10 min.
  • 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.
  • N-ethylmaleimide 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.
  • 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 ⁇ g 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.
  • Liver tissue from rat which remain frozen after removal in liquid nitrogen and stored at -70 0 C, is used for this 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.
  • DMSO non-cooled
  • 1 ml of the solution are used directly and on the other hand 950 ⁇ l of the solution mixed with 50 ⁇ l of 1 M N-ethylmaleimide to a final concentration of 50 mM NEM (solutions used see Table 3).
  • the RNA is isolated from the stored samples.
  • 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.
  • the lysates are centrifuged at 14000 rpm for 3 min. From the supernatant, two portions of 350 ⁇ l each, corresponding to 10 mg of tissue, are removed.
  • 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.
  • 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).
  • 40 ⁇ l of RNase-free water are pipetted onto the membrane to detach the purified RNA from the membrane.
  • 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.
  • Table 3 shows that N-ethylmaleimide also has a positive influence on the stabilization of RNA in biological samples in transition solutions.
  • Liver tissue from rat which was frozen after removal in liquid nitrogen and stored at -70 0 C, is used for this 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.
  • 1 ml of the solution is used directly for the transition and, on the other hand, 950 ⁇ l of the solution are mixed with 100 ⁇ l of IM N-ethylmaleimide (for solutions, see Table 4).
  • 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 also shows that N-ethylmaleimide in transition solutions has a positive influence on the stabilization of RNA in biological samples.
  • Kidney tissue from rat which was frozen after removal in liquid nitrogen and stored at -70 0 C, is used for this 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.
  • 1 ml of the solution is mixed with 50 ⁇ l of 1 M N-ethylmaleimide (solutions used, see Table 5).
  • 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 also shows that the addition of N-ethylmaleimide in the transition solutions described here improves the stabilization of RNA in biological samples.
  • RNA is analyzed on agarose gels stained with ethidium bromide.
  • ethidium bromide 1.0% formaldehyde-agarose MOPS gel is prepared for this purpose. In each case 5 .mu.l of the eluate are used.
  • FIG. From this Figure 2 it can be seen that N-ethylmaleimide also has a positive influence on the quality of the RNA.
  • Liver tissue from rat which remain frozen after removal in liquid nitrogen and stored at -70 0 C, is used for this experiment.
  • 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.
  • 0.5 ml of 100% DMSO is used as a control.
  • 0.5 ml each of a solution of DMSO and the various additives dissolved in A. dest are prepared (for solutions, see Table 6).
  • 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 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):
  • Rat liver tissue that was frozen after removal in liquid nitrogen and stored at -70 0 C, is used for this experiment.
  • 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.
  • 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.
  • 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.
  • 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.
  • Rat liver tissue that was frozen after removal in liquid nitrogen and stored at -70 0 C, is used for this experiment.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un procédé de traitement d'un échantillon biologique, qui consiste à préparer un échantillon biologique et à mettre en contact cet échantillon biologique avec l'acide butènedioïque ou avec un dérivé de l'acide butènedioïque. L'invention concerne également l'échantillon biologique traité pouvant être obtenu au moyen dudit procédé, l'utilisation de l'acide butènedioïque ou de ses dérivés, un échantillon biologique traité ainsi qu'une trousse.
PCT/EP2008/056639 2007-05-31 2008-05-29 Acide butènedioïque ou ses dérivés pour le traitement d'un échantillon biologique WO2008145705A1 (fr)

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US9505776B2 (en) 2013-03-14 2016-11-29 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US9604922B2 (en) 2014-02-24 2017-03-28 Alkermes Pharma Ireland Limited Sulfonamide and sulfinamide prodrugs of fumarates and their use in treating various diseases

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DE102022134188B3 (de) 2022-12-20 2024-03-28 Universität Augsburg - Körperschaft des öffentlichen Rechts Verfahren zur in-situ Erfassung von Änderungen eines Lipidsystems bei dessen Lagerung bei einer Lagertemperatur unterhalb von -60 °C

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US8669281B1 (en) 2013-03-14 2014-03-11 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US9090558B2 (en) 2013-03-14 2015-07-28 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US9505776B2 (en) 2013-03-14 2016-11-29 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US10406133B2 (en) 2013-03-14 2019-09-10 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US10596140B2 (en) 2013-03-14 2020-03-24 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US11083703B2 (en) 2013-03-14 2021-08-10 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US11230548B2 (en) 2013-03-14 2022-01-25 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US11679092B2 (en) 2013-03-14 2023-06-20 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US11905298B2 (en) 2013-03-14 2024-02-20 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US12076306B2 (en) 2013-03-14 2024-09-03 Alkermes Pharma Ireland Limited Prodrugs of fumarates and their use in treating various diseases
US9604922B2 (en) 2014-02-24 2017-03-28 Alkermes Pharma Ireland Limited Sulfonamide and sulfinamide prodrugs of fumarates and their use in treating various diseases

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