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WO2008145708A2 - Alcools aromatiques pour le traitement d'un échantillon biologique - Google Patents

Alcools aromatiques pour le traitement d'un échantillon biologique Download PDF

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Publication number
WO2008145708A2
WO2008145708A2 PCT/EP2008/056644 EP2008056644W WO2008145708A2 WO 2008145708 A2 WO2008145708 A2 WO 2008145708A2 EP 2008056644 W EP2008056644 W EP 2008056644W WO 2008145708 A2 WO2008145708 A2 WO 2008145708A2
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WIPO (PCT)
Prior art keywords
biological sample
composition
weight
samples
frozen
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PCT/EP2008/056644
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German (de)
English (en)
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WO2008145708A3 (fr
Inventor
Vera HOLLÄNDER
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Qiagen Gmbh
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Publication of WO2008145708A2 publication Critical patent/WO2008145708A2/fr
Publication of WO2008145708A3 publication Critical patent/WO2008145708A3/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
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to a method for the treatment, in particular for the stabilization and / or preservation of a biological sample, the treated biological sample obtainable by this method, the use of an aromatic alcohol, the use of a composition 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 agriculture in the cultivation of crops and Livestock, in forensics, in environmental analysis and in many other research areas.
  • RNA patterns transcription patterns
  • PCR real-time reverse transcriptase PCR
  • gene expression chip analyzes, for example, enable recognition of incorrectly expressed genes Genes, which cause, for example, metabolic diseases th, infections or the development of cancer can be detected.
  • Analysis of the DNA from cells by molecular biological methods such as PCR, RFLP, AFLP or by sequencing allows, for example, the detection of genetic defects or the determination of the HLA type and other genetic markers.
  • the analysis of 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 the synthesis of new mRNA molecules, which also greatly alter the transcript pattern of the sample. Therefore, it is necessary to perform an immediate stabilization of the sample to obtain the gene expression profile to be examined. The same applies if a frozen biological sample is used for the purpose of thawed. 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 not only has to unfold on the surface of the cells or within a cell layer, but must also act deep within the multilayered sample material.
  • tissue and / or cell types frequently 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 with regard to the proteins, the carbohydrates and / or the fat content, differ.
  • fixation or stabilization reagents or methods for fixation or stabilization have been developed. wound to stabilize a wide range of different biological samples.
  • the freezing of biological samples has long been known.
  • the sample is immediately after removal from their natural environment at -80 0 C or lower, z. B. in liquid nitrogen, 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 as described in US 6,204,375.
  • the disadvantage of the stabilization processes known from the prior art is that either the stabilization of biomolecules such as nucleic acids in the biological samples is unsatisfactory and, in particular, the yield of these biomolecules from the conventionally stabilized samples is often only low and / or Morphology of the samples is not sufficiently preserved.
  • this stabilization process requires very complicated, logistical preconditions, since thawing of the samples during transport, storage or during a wide variety of application or use processes must be prevented.
  • the use of liquid nitrogen is not only very cumbersome, but only under special precautions feasible.
  • transition solutions are known from the prior art.
  • 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 from -4 ° C. to room temperature only for 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.
  • the object of the present invention was to provide a method for the treatment, in particular for the stabilization and / or preservation, of a biological sample, preferably for the stabilization of a biological sample, with which biomolecules, such as nucleic acids, proteins and metabolites, in which biological sample can be better stabilized.
  • biomolecules such as nucleic acids, proteins and metabolites
  • the object of the present invention was to provide a method for the treatment, in particular for the stabilization and / or preservation, of a biological sample, preferably for the stabilization of a biological sample, with which biomolecules, such as nucleic acids, proteins and metabolites, in which biological sample can be better stabilized.
  • biomolecules such as nucleic acids, proteins and metabolites
  • 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, modification, induction or change in the activity of biomolecules.
  • stabilization should be used.
  • the prevention of a significant change in the morphology of the samples are understood.
  • composition comprising
  • the solvent other than an aromatic alcohol may preferably be an organic solvent.
  • compositions comprising at least one aromatic alcohol as an additive, biomolecules, such as nucleic acids, proteins or metabolites, can be better stabilized in the biological sample.
  • the aromatic alcohol compositions are superior to the corresponding compositions without the aromatic alcohol a better stabilizing ability of biomolecules in a biological sample, in particular by an improved stabilization capacity for nucleic acids.
  • 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 primers, probes or standards used for the PCR, nucleic acids labeled with digoxigenin, biotin or fluorescent dyes or so-called peptide nucleic acids, natural, preferably isolated proteins or oligopeptides, synthetic or modified proteins or oligopeptides For example, antibodies coupled with fluorescent labels or enzymes, hormones, metabolites and metabolites, growth factors, lipids, oligosaccharides, polysaccharides
  • a fresh biological sample is preferably understood to be a sample which, before it is brought into contact with the composition described above in process step ii), is not more than 96 hours, preferably not more than 48 hours preferably not more than 24 hours, moreover preferably not more than 10 hours, moreover 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 before being brought into contact with the composition, for example with conventional fixatives such as formalin.
  • the preparation of fresh cell or tissue samples can be carried out by all the skilled person for this purpose preparation method, in the case of a tissue sample, for example by means of a scalpel, such as surgery or autopsy, in the case of a blood cell sample by centrifugation of fresh blood and the like.
  • a fresh biological Sample serves the composition containing the aromatic alcohol as an additive primarily as a stabilizing composition.
  • method step i) of the method according to the invention preferably uses a biological sample which, after having been isolated, for example, in the manner described above, prior to contacting with the composition described above in method step ii ) are initially at a temperature of 0 0 C or less, preferably to temperatures from -20 0 C or less, and most forthcoming Trains t to temperatures from -70 0 C or less, for example by bringing into contact with liquid nitrogen, was cooled , If a biological sample frozen in this way is used in the process according to the invention, the composition comprising the aromatic alcohol as an additive serves primarily as a transition reagent or as a transition composition.
  • the biological sample is a sample containing cells, such as tissue
  • this biological sample be in the un-lysed state.
  • un-lysed it is meant that the cells have not been lysed by mechanical stress, by treatment by proteases, or by contact with reagents which result in the lysis of cells, for example, by contact with detergents. Any pretreatment of the sample, such as lysis, prior to stabilization would lead to a time delay during which the biomolecule profile of the biological samples is not conserved and thus can be altered.
  • the biological sample having the above-described composition comprising at least one aromatic alcohol, min. at least one solvent other than this aromatic alcohol and optionally one or more additives are brought into contact.
  • the aromatic alcohol (a) is preferably one or more phenol derivatives.
  • Phenol, benzyl alcohol (phenylmethanol), 1, 2-dihydroxibenzol (brenzcachite), 1,3-dihydroxybenzene (resorcinol), 1, 4-dihydroxibenzol (hydroquinone), 1, 4-naphthohydroquinone belong to this group in particular.
  • 2,4,6-trinitrophenol (picric acid), primary phenylethyl alcohol, secondary phenylethyl alcohol, phenylpropyl alcohol, o-tolyl alcohol, p-tolyl alcohol, cumene alcohol, 4-hydroxybenzoic acid, 4-hydroxysulfonic acid, p-nitrophenol, m-, o- or p Alkylphenol, preferably m-, o- or p-methylphenol (cresol) or m-, o- or p-ethylphenol, m-, o- or p-halophenol, preferably m-, o- or p-chlorophenol or m- , o- or p-bromophenol, or mixtures of at least two of these substances. Phenol is the most preferred of all of these.
  • the solvent (b) may be any solvent, including solvents, which may be commonly used for the treatment, in particular for the stabilization of biological samples.
  • the solvent is a solvent selected from the group consisting of water, monohydric, non-aromatic alcohols (monools), polyhydric, non-aromatic 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 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-pentane-triol, 1,2,4-butanetriol, ethylene glycol, propylene glycol, 1,2,3-propanetriol, 1,2,6-hexanetriol, 3-methyl-1,3,5-pentanetriol, acetonitrile, Acetone, anisole, benzonitrile,
  • the additive (c), 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, 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 inhibit 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, lithium nitrate, sodium acetate, potassium acetate, sodium chloride, potassium chloride or calcium chloride
  • osmotically active substances such
  • RNAsecure ® (Ambion) or DEPC
  • alkylating agents alkylating agents, acetylating, halogenation intermediates, nucleotides, nucleotide analogues, amino acids, amino acid analogues, betaine, viscosity regulators, dyes, in particular dyes for the specific staining of specific cell structures, buffer compounds, for example MES, HEPES, MOPS or TRIS, preservatives, complexing agents, such as EDTA or EGTA, Reducing agents, such as, for example, 2-mercaptoethanol, dithiothreitol (DTT), pteridine hydrosulfide, ascorbic acid, NADPH, tricarboxyethylphosphine (TCEP) and hexamethylphosphoric triamide (Me2N) 3P, oxidizing agents, such as 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), substances which improve the permeability of cells,
  • the amount of water should be kept so low that the composition does not lysing when in contact with a biological sample, in particular with a cell-containing biological sample.
  • the aromatic alcohol is phenol
  • the amount of water in the composition is less than 50% by weight, more preferably less than 40% by weight, even more preferably less than 30% % By weight, more preferably less than 20% by weight, even more preferably less than 10% by weight, and most preferably less than 5% by weight, based in each case on the total weight of the composition.
  • the preparation of the composition from the solvent, the aromatic alcohol and optionally the additive is preferably carried out by simple 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.
  • a solid additive may be dissolved in a liquid mixture of solvent and aromatic phenol or solid phenol in a liquid solvent.
  • the contacting of the composition with the biological sample in method 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 can be impregnated by the composition.
  • a biological sample a fluid or isolated cells or z.
  • a granular sample it is contacted by mixing the biological sample with the composition or suspending the biological sample in the composition.
  • the bringing into contact the biological sample with composition 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 moreover 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, -1 ° C, 0 0 C, 1 ° C, 2 ° C, 3 ° C, 4
  • the wording that "contacting the biological sample with composition at a temperature in the range from -80 0 C to +80 0 C” or is effected at one of the other above-mentioned temperatures that bringing into contact according to the the biological sample, the temperature of the mixture thus obtained within the above-mentioned temperatures with the composition for example, it may be 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 composition at a temperature is used that after contacting the frozen biological sample with the composition, the temperature of the mixture (and thus the temperature of the biological sample) in the above mentioned temperature range is.
  • the biological sample may also be preferred for the biological sample to be in contact with the composition in process step ii), preferably under the abovementioned temperature conditions, or in a process subsequent to process step ii) step iii) 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 moreover 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, -11 ° C, -10 0 C, -9 ° C, -8 ° C, -TC, -6 ° C
  • the method of the present invention allows for the storage of a treated biological sample at refrigerator temperatures, at room temperature or at even higher temperatures, without causing any detectable 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 samples can of course, as is common practice, even at low temperatures, for example at -20 0 C or -8O 0 C, be 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 from the biological sample more suitable for histological examinations.
  • suitable embedding means for example in paraffin or the like, in order then to be able to make tissue sections from the biological sample more suitable for histological examinations.
  • this method step iv) may optionally be carried out before or after storage according to the 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 optionally using staining 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, substrates of metabolism, 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 complement DNA (cDNA), counterstrand RNA (aRNA), or synthetic nucleic acids.
  • mRNA messenger RNA
  • 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 RNA-induced silencing complex
  • 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 composition means that the analysis can be done both in situ and ex situ, eg after isolation of the biomolecules from the biological sample it can be isolated from a biological sample for the purpose of analysis be advantageous, especially in the case of cells, tissues or other complex or compact samples to homogenize the samples first, this homogenization by mechanical means, for example by means of cannulas, mortars, rotor-stator homogenizers, a ball mill or the like, by chemical means the use of suitable lysis buffer, which usually include detergents and / or chaotropic substances, can be carried out enzymatically, for example using proteases, or by a combination of these measures.
  • a further contribution to achieving the object stated at the outset is also provided by the use of an aromatic alcohol, preferably of phenol derivatives, more preferably of phenol, in a composition for treating a frozen or unfrozen biological sample according to the method described above.
  • aromatic alcohol preferably of phenol derivatives, more preferably of phenol
  • aromatic alcohols and biological samples of those aromatic alcohols or biological samples are preferred, which have already been mentioned in the beginning in connection with the inventive method.
  • RNAs for the treatment of a biological sample
  • biomolecules such as nucleic acids, proteins or metabolites
  • more preferably of nucleic acids and proteins most preferably of nucleic acids and especially preferred of RNAs, such as mRNAs
  • solvents, aromatic alcohols and additives preference being given as solvents, aromatic alcohols and additives to those compounds which have already been described as preferred embodiments in connection with the process according to the invention.
  • the reagents for analyzing biomolecules in or from a biological sample or for analyzing the morphology of a biological sample may be basically all reagents known to those skilled in the art, which can be used for or in the morphological analysis of a biological sample or for or in the analysis of biomolecules in or from a biological sample.
  • 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.
  • kits according to the invention as a component, for example, at least one device (c), for example in the form of a sealable vessel, for collecting or receiving a solid or liquid biological sample.
  • the device (c) may optionally contain the composition before collection or uptake of the biological sample.
  • the device may be any vessel known to those skilled in the art for collecting or receiving a solid or liquid biological 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 device for the collection or uptake of a solid or liquid biological sample the device optionally comprising, prior to collection or uptake of the biological sample, at least one of the (a) compounds or a composition comprising at least one compound mentioned under (a) may contain.
  • kit according to the invention may contain the above-described components (a), (b) and (c).
  • FIG. 1 shows the Western blot obtained in example 3.
  • FIG. 2 shows a 1% formaldehyde-agarose MOPS gel in which the samples from Example 6 were applied.
  • FIG. 3 shows a 1% formaldehyde-agarose MOPS gel in which the samples from example 8 were applied.
  • FIG. 4 shows a 1% formaldehyde-agarose MOPS gel in which the samples from Example 5 were applied.
  • Kidney tissue of the rat was immediately after organ removal with 500 ul 30% phenol in DMSO added and stored at different temperatures (see Table 1). Following storage, the RNA is isolated from the stored samples.
  • RNA isolation the tissue is removed after storage from the solutions and per 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 remains bound to the membrane and is subsequently washed with a first commercial guanidinium isothiocyanate-containing wash buffer, for example with the buffer RWI from QIAGEN.
  • RNA For elution, 50 ⁇ l 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 total isolated RN A 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 at 280 nm.
  • the results of the insulations are shown in Table 1.
  • RNA is stabilized in biological samples by a composition according to the invention. 2. Stabilization of DNA in biological samples in the presence of phenol
  • Kidney tissue of the rat was immediately after organ removal with 500 ul of 30% phenol in DMSO added 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 manufacturer QIAGEN
  • the lysates are incubated for 2 hours at 55 ° 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 for 10 min at 6000 rpm by centrifugation.
  • 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 in each case passed through the membrane by centrifugation (5 min at 6000 rpm). Subsequently, the plate is incubated for 10 min at 70 0 C.
  • the elution of the DNA is carried out by application of 200 ⁇ l of the at 70 0 C warmed elution buffer AE (QIAGEN). After a one minute incubation, 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.
  • Rat liver tissue was immediately after removal of organs with 1 ml of 30% phenol in DMSO (Probel) or PBS (as a negative control, sample 2) and stored for 3 days at 25 0 C in the incubator. Following storage, a protein extract is prepared from the stored samples. For positive control (sample 3) liver tissue is used, which after removal from the rat frozen directly in liquid nitrogen and then stored at -80 0 C.
  • the tissue is removed from the solutions after storage and 10 mg of tissue, 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, and the sample by means 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 method 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 prior art and with an ERK2-specific antibody, eg. B. the company QIAGEN, hybridized according to the manufacturer, and carried out an immunodetection. The results are shown in FIG.
  • a specific protein in this case a kinase, demonstrates that the proteins are stabilized by the phenol-containing solution at room temperature.
  • Liver and kidney tissue of the rat was immediately after organ removal with 1 ml of 30% phenol dissolved in DMSO and stored for 1 day at 25 ° C in the incubator. After storage: the tissue pieces are removed from the solutions, transferred to plastic cassettes and incubated according to standard protocols in an ascending ethanol series, as well as in xylene and embedded in paraffin. Using a microtome, sections are made from the paraffin-embedded tissue and subjected to hematoxylin-eosin staining on the slide by conventional methods. The stained tissue sections are examined by light microscopy, whereby it can be seen that the solution makes it possible to preserve the morphology of the tissue.
  • 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) treatment compositions (see Table 3) are added and stored for 3 days at 25 0 C.
  • 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, 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, two portions of 350 ⁇ l each, corresponding to 10 mg of tissue, are removed.
  • RNA remains bound to the membrane and is subsequently treated with a first commercially available guanidinium isothiocyanate-containing washing buffer, for example with the buffer RWI from QIAGEN, and then with a second Tris-containing or tris and alcohol-containing washing buffer, eg. B. Buffer RPE from QIAGEN, washed.
  • a first commercially available guanidinium isothiocyanate-containing washing buffer for example with the buffer RWI from QIAGEN
  • a second Tris-containing or tris and alcohol-containing washing buffer eg. B. Buffer RPE from QIAGEN
  • the wash buffers are in each case 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 again for the purpose of complete elution.
  • RNA is analyzed on a 1.0% formaldehyde-agarose MOPS gel stained with ethidium bromide. The result is shown in FIG.
  • Table 3 and FIG. 4 show that phenol is also present in transit compositions has a positive influence on the stabilization of biomolecules such as RNA in biological samples.
  • Liver or kidney tissue of the rat which was frozen after removal in liquid nitrogen and stored at -70 0 C, is for this experiment verwen- det.
  • 20 to 50 mg of tissue are weighed and frozen with various non-cooled treatment compositions (solutions used, see Table 4) and stored at 25 ° C. for 3 days.
  • the RNA is isolated from the stored samples as described in Example 5.
  • 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.
  • phenol has various organic properties
  • Solvents also have a positive influence on the stabilization of RNA in a transition composition.
  • Rat liver tissue which froze 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 mixed with 1 ml each of a non-cooled solution of 25% phenol and 75% DMSO and various additives (solutions used, see Table 5).
  • 950 .mu.l of the phenol-DMSO solution are mixed with 50 .mu.l of each 1 M additives, so that the final concentration of the additives is 50 mM each.
  • DTT as an additive
  • 944.5 ⁇ l of the phenol-DMSO solution are mixed with 55.5 ⁇ l of a 0.9 M DTT solution, so that here too the final additive concentration is 50 mM.
  • 1 ml of the phenol-DSMO solution is used without addition of additive.
  • the samples are stored for 3 days at 25 0 C. Following the transition and storage, the RNA is isolated from the stored samples as described in Example 5. The result is also shown in Table 5.
  • Table 5 also shows that the stabilizing properties of phenol can also be further improved in transition compositions by the addition of certain additives.
  • Liver tissue of the rat which frozen in liquid nitrogen after removal and stored at -70 0 C, is used for this experiment.
  • 20 to 50 mg of tissue are weighed and frozen with a solution of 30% phenol, dissolved in DMSO, pre-cooled to 2 ° C to 8 ° C in the refrigerator.
  • the samples are stored overnight at 2 ° -8 ° C in the refrigerator.
  • the samples are removed from the transition composition and stored 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 is isolated as described in Example 1.
  • the isolated RNA is analyzed on agarose gels stained with ethidium bromide as described in Example 6. The result is shown in FIG.
  • Rat liver tissue was immediately imbibed with ImI of various compositions (see Table 6) after organ removal and stored in the refrigerator at 2-8 ° C for 3 days. Following storage, the RNA is isolated from the stored samples as described in Example 5. The result is also shown in Table 6.

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Abstract

L'invention concerne un procédé destiné au traitement, en particulier à la stabilisation et / ou à la conservation d'un échantillon biologique, comprenant les étapes de procédé suivantes : i) mise à disposition d'un échantillon biologique, et ii) mise en contact de l'échantillon biologique avec une composition de (a) 1 à 90 % en poids au moins d'un alcool aromatique, de (b) 10 à 99 % en poids au moins d'un solvant différent d'un alcool aromatique ainsi que de (c) 0 à 89 % en poids au moins d'un additif différent d'un des composants (a) et (b), tandis que la quantité totale des composants (a) à (c) s'élève à 100 % en poids. L'invention concerne également un procédé destiné au traitement, et en particulier à la stabilisation et / ou à la conservation d'un échantillon biologique, l'échantillon biologique traité et fabriqué par ce procédé, l'utilisation d'un alcool aromatique, l'utilisation d'une composition ainsi qu'un lot.
PCT/EP2008/056644 2007-05-31 2008-05-29 Alcools aromatiques pour le traitement d'un échantillon biologique WO2008145708A2 (fr)

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US5300545A (en) 1987-08-12 1994-04-05 Elf Atochem North America, Inc. Process for stabilizing polymer compositions against heat or light effects
US6204375B1 (en) 1998-07-31 2001-03-20 Ambion, Inc. Methods and reagents for preserving RNA in cell and tissue samples
US6258583B1 (en) * 1999-09-10 2001-07-10 New England Biolabs, Inc. Type II restriction endonuclease, Hpy188I, obtainable from helicobacter pylori J188 and a process for producing the same
DE10031236A1 (de) 2000-06-27 2002-01-10 Qiagen Gmbh Verwendung von Carbonsäuren und anderen Additiven in Kombination mit kationischen Verbindungen zur Stabilisierung von Nukleinsäuren in biologischen Materialien
US6602718B1 (en) 2000-11-08 2003-08-05 Becton, Dickinson And Company Method and device for collecting and stabilizing a biological sample
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US6899850B2 (en) 2001-10-12 2005-05-31 Becton, Dickinson And Company Method and basket apparatus for transporting biological samples
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US20050160701A1 (en) 2003-12-24 2005-07-28 Stevens Timothy A. Tissue preservation assembly and method of making and using the same
US7518035B2 (en) * 2004-03-23 2009-04-14 Monsanto Technology Llc Promoter molecules for use in plants

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