EP1963504A1

EP1963504A1 - Method for the extraction of biomolecules from fixed tissues

Info

Publication number: EP1963504A1
Application number: EP06830682A
Authority: EP
Inventors: Martin Schlumpberger; Ralf Peist
Original assignee: Qiagen GmbH
Current assignee: Qiagen GmbH
Priority date: 2005-12-16
Filing date: 2006-12-18
Publication date: 2008-09-03
Also published as: US20090202998A1; DE102005060738A1; WO2007068764A1; JP2009519027A

Abstract

The invention relates to a method for treating a fixed biological sample, comprising the following steps: i) a fixed biological sample is provided; ii) the fixed biological sample is contacted with an aqueous solution containing at least one nucleophilic reagent; and iii) the biological sample contacted with the aqueous solution is heated. The invention also relates to the biological sample obtained by means of said method, the use of a nucleophilic reagent for treating a fixed biological sample, a kit for isolating a biomolecule from a fixed biological sample, the use of said kit, and a method for treating a disease.

Description

PROCESS FOR EXTRACTION OF BIOMOLECULES FROM FIXED WOVEN FABRICS

The present invention relates to a method for treating a fixed biological sample, the biological sample obtainable by this method, the use of a nucleophilic reagent for treating a fixed biological sample, a kit for isolating a biomolecule from a fixed biological sample, the use of this kit and a method of treating a disease.

If biological material, such as a tissue fragment or isolated cells, is taken from a living organism, they will die after a short time unless suitable measures are taken, such as incubation in nutrient media. The dead cells are very quickly first autolytic fermentative and then bacterially decomposed, so that the original cell and tissue structures are destroyed. If, therefore, cells or tissue fragments are to be removed from an organism for histological examination, it is necessary to fix the removed biological sample in order to prevent its decomposition. This fixation is intended to keep living structures largely life-like, so that they can be assessed in real terms. Depending on the examination objective, the most suitable fixative must be found. The fixation also offers the advantage of being able to store preparations as documents. Therefore, many morphological studies are possible only on the basis of fixed material.

Usually, the fixation is achieved by protein-precipitating or protein-crosslinking compounds such as acids, alcohols, ketones or other organic substances such as glutaraldehyde or formaldehyde, wherein the fixation with formaldehyde (used in the form of a designated as "formalin" 35 wt .-% wässri- QIAGEN GmbH PA180-PCT

solution) followed by embedding of the fixed material in paraffin (so-called "formalin-fixed, paraffin-embedded" (FFPE) material), especially in pathology has the greatest importance.The advantage of formalin fixation over other fixatives, such as 96% denatured alcohol, lies in particular in the fact that the cell and tissue structures in the fixation remain relatively well preserved.

However, the disadvantage of formaldehyde fixation is that, because of the high degree of crosslinking of the biomolecules by the crosslinking formaldehyde, isolation of biomolecules such as DNA, RNA or proteins from a FFPE material is very difficult to isolate them However, biomolecules are of great importance for many studies. Thus, for example, by determining the expression of the enzyme thymidylate synthase in a tumor tissue, a statement can be made as to whether the tumor can be treated successfully with certain cyto-toxic substances or whether the tumor may already have developed resistance to certain cytotoxic agents. If, for example, the mRNA coding for the thymidylate synthase can be isolated from an FFPE tumor tissue, as described in WO-A-01/46402, it is possible to infer the enzyme expression via the amount of mRNA in the tissue and thus the behavior of the tumor against certain cytotoxic agents can be drawn. However, since the biomolecules in the FFPE materials are cross-linked and cross-linked RNA is not a suitable substrate in biochemical assays, particularly in reverse transcription or polymerase chain reaction (PCR), and cross-linked proteins are often very poor antigens for immunological detection, it is required prepare the FFPE material accordingly before analysis of the biomolecules. QIAGEN GmbH PA180-PCT

Thus, WO-A-01/46402 proposes to heat the FFPE material with a chaotropic solution containing an effective amount of a guanidinium compound at a temperature of 75 to 100 ^° C. for 5 to 120 minutes. However, the disadvantage of this method is that the conditions under which the material is heated often leads to at least partial destruction of the biomolecules, in particular sensitive biomolecules such as RNA. Also, the treatment times required for a sufficient solution of formaldehyde crosslinking are often very long.

US-A-2005/0014203 proposes to first heat a fixed biological sample in order to at least partially dissolve the cross-linking of the biomolecules and then to incubate the sample thus treated with a proteolytic enzyme, for example proteinase K, around the tissue and to decompose the cellular structures of the tissue. The disadvantage of this method is likewise that very high temperatures are required for dissolving the crosslinking, especially in the case of short incubation times, and that this process takes place only very slowly at moderate temperatures.

The present invention has for its object to overcome the disadvantages resulting from the prior art.

In particular, the present invention, the object of the invention to provide a method by which a fixed biological sample, preferably fixed with formaldehyde biological sample under the gentlest possible conditions can be treated so that the biomolecules in a simple manner isolated from the biological sample or in the biological sample can be detected. QIAGEN GmbH PA180-PCT

The present invention was also based on the object of specifying a method for treating a preferably fixed with formaldehyde biological sample, with which compared to the known from the prior art method at a treatment temperature of preferably less than 95 ° C, the resulting by fixing Crosslinking in the biological sample can be solved faster.

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

i) providing a fixed biological sample, ii) contacting the fixed biological sample with a preferably aqueous solution containing at least one nucleophilic reagent, and iii) heating the biological solution contacted with the aqueous solution

Sample.

Surprisingly, it was found that the cross-links formed by the fixation of biological samples, in particular by the fixation of formaldehyde, can be dissipated much more quickly within the biological samples in the presence of a nucleophilic reagent.

Furthermore, it was also surprisingly found that in a particular embodiment of the method according to the invention a quantitative analysis of proteins from formalin-fixed tissues is possible. In contrast to the known prior art, the fixed sample is incubated at a temperature between 70 and 90 ⁰ C in the presence of a nucleophilic reagent in the treatment. This leads to a release of a sufficient amount of intact proteins, which are then accurately quantified QIAGEN GmbH PA180-PCT

can. Preferably, the aqueous solution having the fixed biological sample contacted during extraction of proteins does not contain a proteolytically active compound such as a protease.

The fixed biological sample provided in method step i) can be a complete organism, a part of an organism, in particular a tissue fragment or a tissue section, a body fluid, a cell or a virus, cells, tissue fragments and especially tissue sections being particularly preferred biological samples. Again, animals, plants or microorganisms such as bacteria, yeasts or fungi come into consideration as an organism, a human biological sample being a particularly preferred starting material and a human tissue section, a human-isolated cell or a cultured human cell most preferred biological samples.

The fixation of the biological sample can be carried out with all fixatives known to the person skilled in the art, in particular with acids, alcohols, ketones or other organic substances, such as glutaraldehyde or formaldehyde, with formaldehyde-fixed biological samples being particularly preferred and with an aqueous formaldehyde solution, containing 1 to 35 wt .-%, preferably 2 to 10 wt .-% formaldehyde fixed biological samples are most preferred.

According to a particularly preferred embodiment of the method according to the invention, a biological sample fixed in formaldehyde and embedded in paraffin is used as biological sample in method step i). Such samples are commonly referred to as FFPE samples. Such a FFPE sample is preferably prepared by first dewatering a formalin-fixed biological sample, preferably by means of an ascending alcohol series (ie a series of water / alcohol mixtures with increasing amounts). QIAGEN GmbH PA180-PCT

the alcohol concentration, ultimately pure alcohol is added). Cis to Cs alcohols are particularly preferred as alcohols, and ethanol, methanol and isopropanol are moreover preferred and ethanol is most preferred. Subsequently, the dehydrated sample is dipped in liquid paraffin, which is cured after the sample has been sufficiently penetrated by the paraffin. By means of suitable cutting devices, for example by means of a microtome, tissue sections can then be produced from the paraffin block, wherein the thickness of these sections for light microscopic examination is usually about 5 to 20 μm. Furthermore, the paraffined of the sample may also be by other methods, such as by punching with a hollow needle or by the so-called J _^ aser Capture "- procedure into smaller sample fragments are comminuted.

In method step ii), the fixed biological sample provided in method step i) is then brought into contact with a preferably aqueous solution containing at least one nucleophilic reagent. If the biological sample is a paraffin-embedded biological sample, it is preferred to at least partially, preferably completely, remove the paraffin first from the biological sample prior to contacting the sample with the preferably aqueous solution. The removal of the paraffin from the biological sample can in principle be carried out by all methods of deparaffinization of biological samples known to the person skilled in the art. Preferably, the deparaffination is accomplished by first contacting the sample with a hydrophobic organic solvent, especially with an aromatic hydrocarbon, most preferably with xylene, to dissolve out the paraffin. It may also be advantageous to move the mixture of the biological sample and the organic solvent, for example, to shake on a laboratory shaker to ensure the most effective possible dissolution of the paraffin. Advantageously, the mixture is then centrifuged QIAGEN GmbH PA180-PCT

and the organic solution separated from the pellet (= biological sample). Optionally, this step of separating the paraffin from the biological sample may be repeated once, twice or even ten times. In addition to the dissolution of the paraffin by means of a suitable organic solvent, other processes, such as, for example, the melting of the paraffin, can be used as methods for the deparaffinization, as described by Banerjee et al., Biotechniques, 18 (1995), pages 768-773.

After removal of the paraffin, it may be preferable to rehydrate the biological sample, this rehydration is preferably carried out by the stepwise washing with aqueous alcohol solutions with decreasing alcohol concentration (= descending alcohol series), again Ci- to Cs-alcohols are particularly preferred In addition, ethanol, methanol and isopropanol are more preferred and ethanol is most preferred. According to a preferred embodiment of the process according to the invention, an alcohol series having a concentration range from 100% by volume to 70% by volume is used, the concentration difference between two aqueous alcohol solutions successive in their concentration preferably being less than 10% by volume, particularly preferably not more than 5% vol. A descending alcohol series suitable according to the invention comprises, for example, the concentrations 100% by volume, 95% by volume, 90% by volume, 80% by volume and 70% by volume.

In principle, it is also conceivable to carry out the deparaffinization and the rehydration with a single reagent, for example with the commercially available product EZ-DEWAX® from BioGenex, California, USA.

Before the optionally deparaffinized and rehydrated biological sample in process step ii) is brought into contact with the preferably aqueous solution containing the nucleophilic reagent, it may furthermore be advantageous to QIAGEN GmbH PA180-PCT

to dry the sample beforehand, for example by leaving it in air or incubating in a drying oven.

Furthermore, it may be preferred according to the invention to homogenize the optionally deparaffinized and rehydrated biological sample prior to contacting it with the preferably aqueous solution in method step ii), which may be advantageous, in particular for larger tissue fragments. This homogenization can be carried out by any apparatus known to a person skilled in the art for comminuting a biological sample, in particular by high-pressure cell disruption, by means of a mechanical comminution device, for example a mill, a rotor-stator homogenizer, an Ultra-Turrax homogenizer or a fine cannula, or by ultrasonic homogenizers.

In method step ii) of the method according to the invention, the fixed biological sample is then brought into contact with a preferably aqueous solution containing at least one nucleophilic reagent.

As nucleophilic reagent all Lewis bases are suitable, which are able to transfer electrons into an empty orbital or in empty orbitals of a Lewis acid. Particularly preferred among these Lewis bases are reagents which have at least one functional group which carries a negative charge which is negatively polarized or which has at least one free electron pair.

Compounds comprising a functional group having a negative charge include, for example, alkali or alkaline earth alkoxides, alkali or alkaline earth hydroxides, alkali or alkaline earth halides, alkali or alkaline earth cyanides, and the like. QIAGEN GmbH PA180-PCT

Reagents which have at least one functional group which is negatively polarized are, in particular, those reagents which have at least one functional group in which there are two covalently linked atoms which in their electronegativity according to Alfred and Rochow are at least 0, 25, more preferably by at least 0.5 and more preferably by at least 1, 0 differ.

However, nucleophilic reagents which are particularly preferred according to the invention are those which have at least one functional group having one or two, more preferably a lone pair of electrons, of which in turn those are most preferred which contain at least one primary, secondary or tertiary amino group. Group of structure I

2

RN o ^S R ³ I, in the

R ^{1 is} a Ci to C ₂ o hydrocarbon group, more preferably a C ₂ - to Ci5 hydrocarbon group and more preferably a C ₂ to Cio hydrocarbon group having at least one heteroatom

C 1 to C ₂ o-hydrocarbon group, a C ₂ - to Cis-hydrocarbon group having at least one heteroatom and moreover preferably a C ₂ - to C ₁₀ -hydrocarbon group having at least one heteroatom or an optionally heteroatom-substituted is aromatic ring system, QIAGEN GmbH PA180-PCT

R ^{2 is} a C 1 to C 20 alkyl group, particularly preferably a C 1 to C ₁₀ alkyl group and moreover preferably a C 1 to C 2 alkyl group, in particular a methyl group or an ethyl group, a C 1 to C 20 hydroxyalkyl group , particularly preferably a C 1 to C ₁₀ -hydroxyalkyl group and moreover preferably a C 1 to C ₂

Hydroxyalkyl group or a hydrogen atom, with a hydrogen atom being most preferred, and

R ^{3 is} a C 1 to C 20 alkyl group, particularly preferably a C 1 to C ₁₀ alkyl group and moreover preferably a C 1 to C 2 alkyl group, in particular a methyl group or an ethyl group, a C 1 to C 20

Hydroxyalkyl group, particularly preferably a C 1 to C ₁₀ -

Hydroxyalkyl group and moreover preferably a C 1 to C ₂ -

Hydroxyalkyl group or a hydrogen atom, with one hydrogen atom being most preferred.

Especially preferred nucleophilic reagents having a functional group of structure I shown above are those which have at least one functional group of structure I in which at least one of R ² and R ³ , most preferably both R ² and R ³ is a hydrogen atom or are. In addition, particularly those nucleophilic reagents are preferred which have at least one functional group of the structure I, in which the nitrogen atom is covalently linked only to those atoms in the radicals R ¹ , R ² and R ³ , which are sp ³ hybridized. In particular, none of the radicals R ¹ , R ² or R ^{3 should be} able to delocalize the free electron pair on the nitrogen atom over the radicals R ¹ , R ² or R ³ . Thus, it is particularly preferred that none of the radicals R ¹ , R ² and R ^3, for example, the structure II QIAGEN GmbH PA180-PCT

H

IN H

- C-N

H

II

having.

According to the invention, particularly preferred nucleophilic reagents having at least one functional group of the structure I are selected from the group consisting of methylamine, ethylamine, ethanolamine, n-propylamine, n-butylamine, isobutylamine, tert-butylamine, dimethylamine, diethylamine, diethanolamine, di-methylamine. n-propylamine, di-iso-propylamine, dibutylamine, trimethylamine, triethylamine, triethanolamine, hexamethylenetetramine, 2-ethylhexylamine, 2-amino-l, 3-propanediol, hexylamine, cyclohexylamine, 1, 2-di-methoxypropanamine, 1-aminopentane , 2-methyloxypropylamine, tri (hydroxymethyl) aminomethane, aminocarboxylic acids, especially glycine or histidine, or aminoguanidine, among which ethanolamine, diethanolamine, triethanolamine, amino-1,3-propanediol, aminoguanidine and tri (hydroxymethyl) aminomethane are the most are preferred. Furthermore, as nucleophilic reagents having at least one functional group of structure I, aromatic amines are selected from the group consisting of aniline, toluidine, naphthylamine, benzylamine, xylides, xylenediamines, naphthalenediamines, toluene diamines, 3,3'-dimethyl-4,4 ' diphenyldiamine, phenylenediamines, 2,4'-methylenedianiline, 4,4'-methylenedianiline, sulfonyldianiline, and dimethylbenzylamine.

According to a particular embodiment of the method according to the invention, wherein the nucleophilic reagent is at least one primary amino group of

Structure I, the nucleophilic reagent is a cis bis

C _ό alkylamine, _ό a Ci to Co-alkyl diamine, a Ci to C -Alkyltriamin, QIAGEN GmbH PA180-PCT

a Ci to C ₁₅ aminoalcohol around a Ci to C ₁₅ Aminodiol, or around a Ci to C ₁₅ Aminocarbonsäure.

According to another particular embodiment of the process according to the invention, the nucleophilic reagent is a heterocyclic, nitrogen atom-containing compound selected from the group consisting of pyrrole, pyridine, quinoline, indole, aza-cyclopentane, aza-cyclohexane, morpholine, piperidine, imidazole or a derivative of these compounds, a derivative of these compounds preferably being understood as meaning a derivative in which one to one or more carbon atoms or to the nitrogen atom in the abovementioned compounds contains a C 1 -C 6 -alkyl group, particularly preferably a methyl group or ethyl Group is bound instead of a hydrogen atom.

Of the nucleophilic reagents mentioned above, particular preference is given to those which are water-soluble, in particular those which in water at a temperature of 25 ° C. and at a pH of 7 have a solubility of at least 1 g / L, especially preferably at least 10 g / L and moreover preferably at least 100 g / L.

The preferably aqueous solution comprising the above-described nucleophilic reagent can be based on pure, preferably deionized water or else on other aqueous systems, in particular mixtures of water and organic solvents such as alcohols, in particular mixtures of water and ethanol or methanol, wherein the amount water, preferably at least 50% by weight, more preferably at least 75% by weight and most preferably at least 90% by weight, based in each case on the total weight of water and organic solvent, of physiological saline solutions, on buffers, in particular buffers including buffer components known to those skilled in the art, such as TRIS, HEPES, PIPES, QIAGEN GmbH PA180-PCT

CAPS, CHES, AMP, AMPD or MOPS in an amount ranging from 0.1 to 1000 mmol / l, more preferably 1 to 500 mmol / l and most preferably 10 to 200 mmol / l, optionally containing such a buffer component, depending on their structure, can also serve as a nucleophilic reagent at the same time. Furthermore, also nutrient media, such as MEM medium and DMEM medium can be used as an aqueous system. The preparation of the aqueous solution containing the nucleophilic reagent is preferably carried out by simply mixing water or a corresponding aqueous system with the nucleophilic reagent.

The concentration of the nucleophilic reagent in the aqueous solution is preferably in a range of from 0.1 to 10,000 mmol / l, more preferably from 1 to 5,000 mmol / l, still more preferably from 5 to 2,500 mmol / l, and most preferably from 20 to 1000 mmol / l. According to a particularly advantageous embodiment of the method according to the invention, the concentration of the nucleophilic reagent in the aqueous solution is more than 20 mmol / l, more preferably more than 50 mmol / l and most preferably more than 100 mmol / l.

The pH of the aqueous solution is preferably in a range of from 2 to 12, more preferably from 4 to 9, and most preferably from 5 to 8, each measured at room temperature.

The contacting of the aqueous solution with the biological sample is preferably carried out by immersing the biological sample in a simple manner in a sufficient amount of the aqueous solution. If, for example, the biological sample is in the form of a tissue section and in the form of an adherent cell on a substrate, the contact between the biological QIAGEN GmbH PA180-PCT

gical sample with the aqueous solution, preferably by simply coating the substrate with the aqueous solution.

In process step iii) of the process according to the invention placed with the aqueous solution into contact biological sample is now heated, preferably to a temperature in a range of 50 to 100 ⁰ C, particularly preferably from 55 to 95 ° C, more preferably of 60 is heated to 90 ⁰ C and most preferably 65 to 85 ° C. In particular, it can erfmdungsge- Mäss be advantageous, brought into contact with the aqueous solution of biological see sample to a temperature of less than 95 ° C, more preferably less than 90 ⁰ C, and most preferably less than 80 ⁰ C to heat.

In a particular embodiment of the method according to the invention, which can be used for the analysis of proteins from the fixed sample, in method step iii) the biological sample brought into contact with the aqueous solution is at a temperature in a range from 65 to 85 ° C, especially preferably heated from 75 to 85 ⁰ C.

Preferably, in a particular embodiment of the method according to the invention, which can be used for the analysis of proteins from the fixed sample, the biological sample is boiled for 5 to 40 minutes before process step iii), ie heated to about 100 ^° C.

The duration of heating in step iii) depends essentially on the temperature and the reactivity of the nucleophilic reagent, and the person skilled in the art will be able to ascertain by simple routine experiments when sufficient destruction of the cross-linking of the biological sample has occurred under the given treatment conditions. Usually, the duration of heating is QIAGEN GmbH PA180-PCT

however, in a range of 60 seconds to 10 hours, more preferably 2 minutes to 5 hours, and most preferably in a range of 10 minutes to 2 hours.

In a particular embodiment of the method of the invention that can be used to analyze proteins from the fixed sample, the duration of heating may be up to 16 hours.

Depending on the nature and composition of the biological sample used in process step i), it may also be advantageous, after heating in process step iii), to comminute the biological sample (especially if the sample is not comminuted before treatment with the nucleophilic reagent) In turn, any apparatus known to a person skilled in the art for comminuting biological samples may be used for comminuting. In particular, the comminution can be effected by means of high-pressure cell disruption, by means of a mechanical comminution device, for example a rotor-stator homogenizer, a mill, an Ultra-Turrax homogenizer or a fine cannula, or by ultrasound homogenizers.

According to a particular embodiment of the method according to the invention, the biological sample during the heating in step iii), before step ii) or after step iii) contacted with compounds, preferably incubated, which destruction of a biological tissue and / or the Lysis of cells promotes, wherein this compound is preferably an enzyme, a detergent, a chaotropic substance or a mixture of at least two of these components.

Particularly preferred enzymes in this context are proteases, of which trypsin, proteinase K, chymotrypsin, papain, pepsin, pronase QIAGEN GmbH PA180-PCT

and endoproteinase Lys-C. particularly preferred and proteinase K is most preferred. According to a particular embodiment of the method according to the invention, however, a thermostable protease can also be used as the enzyme, as described for example in WO-A-91/19792 (isolated from Thermococcus celer, Therococcus sp.ANl, Thermococcus stetteri or Thermococcus litoralis) or in WO-A-91/19792 (isolated from Staphylothermus marinus). The disclosure of these references in terms of heat stable proteases is hereby incorporated by reference and forms part of the disclosure of the present invention.

In a particular embodiment of the method according to the invention, which can be used for the analysis of proteins from the fixed sample, no proteolytically active compound such as a protease is used. In a specific embodiment, however, a nuclease such as a DNase and / or RNase, preferably a heat-stable nuclease, may be used here.

The concentration of the enzyme in the aqueous solution is preferably in a range of 0.001 to 5% by weight, more preferably 0.01 to 2.5% by weight, and most preferably 0.05 to 0.2% by weight. , in each case based on the total weight of the aqueous solution.

Preferred detergents are compounds selected from the group comprising sodium dodecyl sulfate (SDS), polyethylene glycol phenol ethers such as Triton-X-100, Tween, NP-40 or mixtures thereof, with SDS and Triton-X-100 being particularly preferred as detergents , The amount of detergent used to lyse the cells contained in the biological sample will depend on the type and amount of biological sample and can be determined by one skilled in the art by simple routine experimentation. QIAGEN GmbH PA180-PCT

Very particular preference is given in a particular embodiment of the method according to the invention which can be used for the analysis of proteins from the fixed sample as a detergent SDS, sodium deoxycholate, CHAPS, Triton X100, Nonidet P40 or Tween 20 used. The concentration of detergent here is preferably in a range between about 0.1-10%, more preferably between 1-5%.

Guanidinium isothiocyanate or guanidinium hydrochloride are particularly preferred as chaotropic substances, with guanidinium isothiocyanate being particularly preferred. Chaotropic substances are used in particular when nucleic acids are to be isolated from the biological sample after the treatment according to the invention. In this context, it is further preferred that in addition to the chaotropic compound, reducing compounds, in particular dithiothreitol (DTT) or ß-mercaptoethanol, are used. The concentration of chaotropic compound in the treatment of the biological sample is preferably in a range of 0.1 to 50 mol / l, more preferably 0.5 to 20 mol / l, and most preferably 1 to 10 mol / l.

The incubation of the biological material with the enzyme, the detergent or the chaotropic compound can, as stated above, before, during the heating in step iii), before the process step ii) or after the process step iii) take place.

According to a particular embodiment of the method according to the invention, the incubation of the biological material with the enzyme, the detergent and / or the chaotropic compound takes place before process step ii). For this purpose, for example, before process step ii), the biological sample with an aqueous solution containing the enzyme, the detergent, the chaotropic compound QIAGEN GmbH PA180-PCT

or at least two thereof, optionally together with a reducing compound such as .beta.-mercaptoethanol, brought into contact in the form of a lysis buffer, heated to a temperature required for a sufficient enzyme activity and then this lysis buffer replaced by the aqueous solution include the nucleophilic reagent or an appropriate amount of nucleophilic reagent added to this lysis buffer.

According to another particular embodiment of the process according to the invention, the incubation of the biological material with the enzyme, the detergent or the chaotropic compound is carried out after process step iii). For this purpose, either after the heating in step iii), the aqueous solution containing the nucleophilic reagent can be removed from the biological sample and this biological sample then contacted with the lysis buffer, or the aqueous solution containing the nucleophilic reagent becomes the enzyme Detergent, the chaotropic compound or at least two thereof, optionally in the form of a concentrated solution added. Subsequently, heating takes place to a temperature necessary for adequate lysis. In particular, when using chaotropic substances, it is preferred that they are added only after the process step iii).

In accordance with a further particular embodiment of the method according to the invention, the incubation of the biological material with the enzyme, the detergent or the chaotropic compound takes place during process step iii). For this purpose, the aqueous solution used in process step ii) is an aqueous solution which, in addition to the nucleophilic reagent, contains the enzyme, the detergent, the chaotropic compound or at least two thereof, or the enzyme is added to the aqueous solution prior to heating in process step iii) , the detergent, the chaotrope compound or at least two thereof, optionally in the form of a concentrated aqueous solution, is added. In the- QIAGEN GmbH PA180-PCT

In this connection, it is particularly preferred to add one of the abovementioned heat-stable proteases or nucleases as the enzyme, since in this way the temperature for a satisfactory solution of the crosslinking is preferably from 50 to ^100.degree. C., more preferably from 55 to 95.degree. even more preferably from 60 to 90 ⁰ C and most preferably 65 to 85 ° C without affecting the enzyme activity can be maintained.

Regardless of the way in which the tissue is treated in steps i) to iii), however, following the heating of the biological sample, an aqueous solution is generally present in which at least part of the biological sample removed by the treatment according to the invention dissolved biomolecules is dissolved. In addition to the liberated biomolecules, this aqueous solution may also comprise other components, such as the nucleophilic reagent, enzymes, detergents, chaotropic compounds, and the like.

According to a particular embodiment of the process according to the invention, this process also comprises the process step in addition to process steps i) to iii)

iv) analysis of a biomolecule dissolved out of the biological sample,

wherein the biomolecule is preferably a protein, a glycoprotein, a lipid, a glycolipid, an RNA or a DNA, most preferably RNA.

In this case, as a starting composition for the analysis of the liberated from the biological sample biomolecule QIAGEN GmbH PA180-PCT

Al) the aqueous solution containing the nucleophilic reagent which was used in process step ii) and in which, after process step iii), part of the biomolecules is present,

A2) the aqueous solution containing an enzyme, a detergent and / or a chaotropic compound, provided that the aqueous solution containing the nucleophilic reagent after step iii) replaced by a corresponding lysis buffer or the aqueous solution containing the nucleophilic reagent before or after step iii ) corresponding lysis components have been added,

A3) an aqueous solution, which after purification of a biomolecule in one of the two aforementioned compositions Al) or A2), for example by filtration, dialysis, extraction, precipitation, chromatography or a combination of these purification methods, but preferably by adsorption obtained In particular, a purification by separation of the aqueous solution Al) or A2) into a protein fraction, an RNA fraction and a DNA fraction, for example by selective precipitation, extraction or adsorption is particularly preferred and a separation by adsorption on most preferred is, or

A4) an aqueous solution which has been obtained by extraction of the tissue obtained after step iii) with a corresponding aqueous phase serve.

The analysis of the biomolecule in the respective compositions A1) to A4) is preferably carried out by the analysis methods known to the person skilled in the art. For example, immunological methods such as Western European QIAGEN GmbH PA180-PCT

tern blotting, enzyme-linked immunosorbent assays (ELISAs), immunoprecipitation or affinity chromatography, mutation analyzes, polyacrylamide gel electrophoresis (PAGE), in particular two-dimensional polyacrylamide gel electrophoresis (2D-P AGE), high performance liquid chromatography (HPLC) , Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) Analysis, Southern Blotting, Serial Analysis of Gene Expression (SAGE), Fast Protein Liquid Chromatography (FPLC), MALDI-TOFF Mass Spectrometry, SELDI Mass Spectrometry, Microarray analysis and the like into consideration.

Depending on the biomolecule to be analyzed, the biological sample may be brought into contact with at least one enzyme during heating in process step iii), before process step ii) or after process step iii). Preferably, prior to process step ii), the biological sample can be brought into contact with at least one enzyme.

When the biomolecule to be analyzed is DNA and / or RNA, the enzyme may be a protease as disclosed above. When the biomolecule to be analyzed is a protein, the enzyme may be a nuclease as disclosed above.

A particular embodiment of the invention comprises a method for treating a formaldehyde-fixed biological sample for later analysis of proteins contained in the sample, comprising the method steps:

i) providing a fixed biological sample, ii) contacting the fixed biological sample with an aqueous solution containing at least one nucleophilic reagent, and QIAGEN GmbH PA180-PCT

iii) heating the biological sample brought into contact with the aqueous solution to a temperature in the range from 65 to 85 ° C; iv) analysis of a biomolecule dissolved out of the biological sample.

In process step ii), a detergent such as SDS is particularly preferably added and / or the detergent is already present in the aqueous solution. The biological sample is preferably boiled in the aqueous solution prior to step iii) for 5 to 40 minutes, and then preferably incubated in step iii) for a period of 20 minutes to 16 hours.

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

Also, the use of a nucleophilic reagent, as described above, in particular a nucleophilic reagent having at least one primary, secondary or tertiary amino group of the structure I, for treatment of a fixed biological sample, in particular for the treatment of a formaldehyde fixed with biological sample affords a contribution to the solution of the aforementioned tasks.

A further contribution to the solution of the abovementioned objects is provided by a kit for isolating a biomolecule from a fixed, preferably from a biological sample fixed with formaldehyde

(αl) a buffer containing a nucleophilic reagent,

(α2) a matrix for the adsorption of a biomolecule, as well QIAGEN GmbH PA180-PCT

(α3) optionally an elution buffer,

(α4) optionally an enzyme, as well

(α5) optionally a chaotropic substance.

Particularly preferred nucleophilic reagents are those reagents which have already been mentioned as preferred reagents in connection with the process according to the invention.

According to a preferred embodiment of the kit according to the invention, it also contains components for lysing a cell, in particular enzymes (α4), preferably one of the proteolytic enzymes mentioned above in connection with the method according to the invention, detergents or chaotropic substances (α5), preferably one of the chaotropic substances mentioned above in connection with the method according to the invention. In particular, the enzyme and the chaotropic substance may be contained, alone or in combination, already in the buffer (α1) containing the nucleophilic reagent. However, it is also possible that the kit comprises a corresponding lysis buffer (αl ') in which these compounds are contained and which can be used for the digestion of a biological sample. Also, the kit according to the invention may comprise a lysing concentrate (αl ") which contains an enzyme, a detergent or a chaotropic compound in concentrated form and which may be added to the buffer (αl) at an appropriate time when applying the kit.

As a matrix (α2) for adsorbing a biomolecule, it is possible to use all materials known to the person skilled in the art for adsorbing a biomolecule, in particular a protein. QIAGEN GmbH PA180-PCT

teins, a DNA or an RNA, using cellulose-based materials, in particular carboxy-functional cellulosic materials or diethylaminoethyl cellulose, agarose, or mineral carriers such as silica, glass, quartz, zeolites, or metal oxides, or coated with ion exchange materials Carrier, are particularly preferred. The materials mentioned can be present for example in the form of membranes or magnetic or non-magnetic particles. Preferably, this matrix is contained as a column material in already prefabricated columns or as a suspension in the kit. The type of matrix depends decisively on the chemical structure of the biomolecules to be analyzed, it being clear to the person skilled in the art for the particular application, eg. As analysis of proteins, RNA or DNA, suitable adsorption materials are known.

As elution buffer, it is likewise possible for all buffers known to the skilled person to be present in the kit according to the invention, which are customarily used for elution in column chromatography. The elution buffer is preferably an aqueous salt solution, in particular aqueous solutions containing alkali halides, such as NaCl, KCl or LiCl, alkaline earth halides, such as CaCl ₂ or MgCl ₂ , ammonium salts such as ammonium chloride or ammonium sulfate or mixtures of at least two optionally, buffering systems such as alkali acetate / acetic acid or tris (hydroxymethyl) aminomethane based buffer systems may also be included in the elution buffer. If the kit is used to isolate RNA from a fixed tissue and a silica membrane is used as the matrix, it is particularly preferred to use water, in particular RNase-free water, as the elution buffer.

According to a further, particular embodiment of the kit according to the invention, the latter may also contain a wash buffer with which the matrix can be detected. QIAGEN GmbH PA180-PCT

to which it has bound the biomolecule to be analyzed, before being eluted with the elution buffer.

A contribution to the solution of the problem mentioned at the outset continues to be provided by the use of the kit according to the invention in a method for isolating biomolecules from a fixed, preferably a biological sample fixed with formaldehyde.

Finally, a method for the treatment of a disease, comprising the method steps, also contributes to the solution of the abovementioned objects:

(β1) removal of a biological sample from an organism, preferably from a mammal, more preferably from a human or from an animal,

(ß2) fixation of the biological sample, preferably with formaldehyde,

(β3) analysis of a biomolecule from the fixed biological sample by the method according to the invention comprising the method step iv),

(ß4) diagnosis of a disease based on the results of the analysis, as well

(ß5) therapeutic treatment of the diagnosed disease.

As biological sample and as biomolecule those biological samples and biomolecules are again preferred, which were already described in the beginning in connection with the method according to invention. QIAGEN GmbH PA180-PCT

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

FIG. 1 shows the yield of RNA (in ng) from a formalin-fixed and deparaffinized tissue section from the lung of a rat after the treatment according to the invention.

FIG. 2 shows the behavior of the RNA (in terms of the number of PCR cycles required to reach a specific DNA amount in a real-time RT-PCR analysis), which is determined by the method according to the invention from a formalin-fixed and deparaffinized tissue section the rat liver was isolated in a PCR analysis.

FIG. 3 shows the yield of RNA (in ng) from a formalin-fixed and deparaffinized tissue section from the liver of a rat after the treatment according to the invention.

Figure 4 shows the yield of intact protein from a formalin-fixed and deparaffinized tissue section from the gut and lung of a rat after treatment according to the invention.

FIG. 5 shows the yield of intact proteins from a formalin-fixed and deparaffinized tissue section from the heart of a rat after the treatment according to the invention as a function of the temperature in the incubation step.

EXAMPLES QIAGEN GmbH PA180-PCT

example 1

Formalin-fixed and deparaffinized tissue sections (10 μm) from the lung of a rat were each treated with 250 μl of an aqueous solution containing 5 mol / l guanidine hydrochloride and different nucleophilic reagents (13 mM TRIS, 350 mM TRIS, 5% by weight). Trimethylamine, 10 mg / ml aminoguanidine or 50 mg / ml aminoguanidine), the pH of the aqueous solutions being 7.5 each. The sections were incubated at 70 ^° C. for 60 minutes with the appropriate solutions.

The RNA was isolated from the obtained cell lysate. For this purpose, initially genomic DNA using the gDNA eliminator Mini spin column (Qiagen, Hilden, Germany), the flow collected, mixed with 400 ul ethanol, and the thus obtained composition to an RNeasy ^® MinElute spin column (Qiagen, Hilden, Germany) applied. After washing twice using the RNeasy ^® kit contained in the RPE buffer, the membrane was dried and the RNA eluted RNase-free by means of 30 ul of water. The amount of RNA in the eluate was determined by OD measurement at 260 nm. The results are shown in FIG.

Example 2

Formalin-fixed and deparaffinized tissue slices (10 μm) from the liver of a rat were incubated with 250 μl of an aqueous solution containing different nucleophilic reagents (10 mM TRIS, 10 mM TRIS and 1% ethanolamine, 10 mM TRIS and O, 1% ethanolamine and 10 mM TRIS and 0.01% ethanolamine), the pH of each of the aqueous solutions being 7.5. The cells were incubated for 60 minutes at 70 ^° C. with the appropriate solutions. Guanidine hydrochloride was then QIAGEN GmbH PA180-PCT

ner final concentration of 5 mol / l added.

The RNA was isolated from the cell lysate obtained and the behavior of the RNA in the PCR was determined by means of a TaqMan analysis using the QuantiTect Probe RT-PCR Kit from Qiagen, Hilden, Germany, and a corresponding primer / probe set. For this purpose, initially genomic DNA using the gDNA eliminator Mini spin column (Qiagen, Hilden, Germany), the flow collected, mixed with 400 ul ethanol, and the thus obtained composition to an RNeasy ^® MinElute spin column (Qiagen, Hilden, Germany) applied. After washing twice by means of the RNeasy ^® - contained Kit RPE buffer, the membrane, the RNA by 30 ul RNase-free water was dried and eluted. The results of the PCR are shown in FIG. In addition to the analysis of the behavior of the RNA in the PCR, the amount of isolated RNA was determined in Example 2 by means of OD measurement at 260 nm. The results are shown in FIG.

FIGS. 2 and 3 show that high amounts of RNA can be isolated by the treatment according to the invention of the samples and that the isolated RNA also shows a good behavior in the PCR analysis.

Example 3

Verification of the temperature range for the quantitative extraction of intact, full-length proteins from formalin-fixed, paraffin-embedded tissue

Formalin-fixed and deparaffinized tissue sections (10 μm) from the gut and brain of a rat were in contact with 250 μl each of an aqueous solution containing nucleophilic reagents 1.25 M Tris / HCl (pH 6.8) and 4% SDS. QIAGEN GmbH PA180-PCT

introduced. The sections were boiled for 20 min at 100 ⁰ C and then incubated for 2 hours at 80 ⁰ C.

The incubation step was carried out using the example of two different tissues at different temperatures. From the lysates obtained, equal volumes were subsequently analyzed in the Western blot. The signal for actin was determined densitometrically using Easy win and compared. The signal of the 80 ⁰ C samples were always set = 100%.

The samples extracted at 80 ^° C showed the strongest signals in the different tissues for both actin and tubulin.

Example 4:

The temperature differences of the second incubation step were chosen smaller in this example (brain, rat). The experiments were carried out analogously to the protocol described in Example 3.

It can clearly be seen that the strongest signals are ren in the samples to be detected, which were incubated in a range of 70 to 85 ⁰ C, especially at 80 ° or 85 ° C.

Claims

QIAGEN GmbH PA180-PCTPATENTANSPRÜCHE

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

i) providing a fixed biological sample, ii) contacting the fixed biological sample with an aqueous solution containing at least one nucleophilic reagent, and iii) heating the biological sample contacted with the aqueous solution.

The method of claim 1, wherein the fixed biological sample is a formaldehyde-fixed biological sample.

The method of claim 2, wherein the formaldehyde-fixed biological sample is a formaldehyde-fixed and paraffin-embedded biological sample.

The method of claim 3, wherein the paraffin is at least partially removed prior to contacting the aqueous solution.

5. The method according to any one of the preceding claims, wherein the nucleophilic reagent is a compound having at least one functional group carrying a negative charge which is negatively polarized or which has at least one lone pair of electrons. QIAGEN GmbH PA180-PCT

6. The method according to any one of the preceding claims, wherein the nucleophilic reagent is a compound which has at least one primary, secondary or tertiary amino group of the structure I.

2

RN o ^S R ³ I, in the

R ¹ is a Ci to _C2o hydrocarbon group, an at least one heteroatom-containing Ci to _C2o hydrocarbon group or an optionally heteroatom substituted aromatic ring system, R ² is a Ci to C ₂ o Alkyl group, a Ci to C ₂ o-hydroxyalkyl group or a hydrogen atom, and

R ^{3 is} a Ci to C ₂ o-alkyl group, a Ci to C ₂ o-hydroxyalkyl group or a hydrogen atom.

7. The method of claim 6, wherein at least one of R ² and R ^{3 is} a hydrogen atom.

8. The method of claim 6 or 7, wherein both R ² and R ^{3 is} a hydrogen atom.

9. The method according to claim any one of claims 5 to 8, wherein the nucleophilic reagent is a Ci to Cό-alkylamine or a Ci to C ₁₅ aminoalkohol, aminodiol or an aminocarboxylic acid. QIAGEN GmbH PA180-PCT

10. The method according to any one of the preceding claims, wherein the nucleophilic reagent is a heterocyclic, a nitrogen atom-containing compound selected from the group comprising pyrrole, pyridine, piperidine, quinoline, indole, aza-cyclopentane, aza-cyclohexane, morpholine or a derivative thereof Links.

11. The method according to any one of the preceding claims, wherein the nucleophilic reagent is selected from the group consisting of ethanolamine, diethanolamine, triethanolamine, amino-l, 3-propanediol, aminoguanidine and tri (hydroxymethyl) amino methane.

12. The method according to any one of the preceding claims, wherein the nucleophilic reagent at a temperature of 25 ° C and at a pH of 7 has a solubility in water of at least 1 g / L.

13. The method according to any one of the preceding claims, wherein the nucleophilic reagent is contained in a concentration in a range of 0.1 to 10,000 mmol 1/1 in the aqueous solution used in step ii).

14. The method according to any one of the preceding claims, wherein in step iii) to a temperature in a range of 50 to 100 ⁰ C is heated.

15. The method according to any one of the preceding claims, wherein in step iii) for a period of time in a range of 60 seconds to 10 hours is heated. QIAGEN GmbH PA180-PCT

16. The method according to any one of the preceding claims, wherein the method in addition to the process steps i) to iii) the process step

iv) Analysis of a biomolecule released from the biological sample

includes.

17. The method of claim 19, wherein the biomolecule is RNA, DNA or a protein.

18. The method of claim 16 or 17, wherein during the heating in step iii), before step ii) or after procedural step iii) the biological sample is brought into contact with at least one enzyme.

The method of claim 18, wherein when the biomolecule is DNA and / or RNA, the enzyme is a protease.

The method of claim 17, wherein when the biomolecule is a protein, the enzyme is a nuclease.

21. A biological sample obtainable by the method according to any one of claims 1 to 20.

22. The use of a nucleophilic reagent as defined in any one of claims 5 to 12 for the treatment of a fixed biological sample.

23. A kit for isolating a biomolecule from a fixed biological sample, comprising QIAGEN GmbH PA180-PCT

(αl) a buffer containing a nucleophilic reagent,

(α2) a matrix for adsorbing a biomolecule,

(α3) optionally an elution buffer,

(α4) optionally an enzyme, as well

(α5) optionally a chaotropic substance.

A kit according to claim 23, wherein the kit comprises apart from the components (α1) to (α2) and optionally (α3) to (α5) a washing buffer (α6).

25. The use of the kit according to claim 23 or 24 in a method for isolating biomolecules from a fixed biological sample.

26. A method of treating a disease, comprising the steps of: (β1) removing a biological sample from an organism,

(β2) fixation of the biological sample with formaldehyde, (β3) analysis of a biomolecule from the formaldehyde-fixed biological sample by the method according to claim 19,

(ß4) diagnosis of a disease based on the results of the analysis, as well as

(ß5) therapeutic treatment of the diagnosed disease. QIAGEN GmbH PA180-PCT

27. A method of treating a formaldehyde-fixed biological sample for later analysis of proteins contained in the sample, comprising the steps of:

i) providing a fixed biological sample, ii) contacting the fixed biological sample with an aqueous solution containing at least one nucleophilic reagent, and iii) heating the biological sample brought into contact with the aqueous solution to a temperature in one Range from 65 to

85 ° C, iv) analysis of a biomolecule dissolved out of the biological sample.

28. The method of claim 27, wherein the aqueous solution in step ii) contains a detergent.

29. A method according to claim 27 or 28, wherein the biological sample is boiled in the aqueous solution prior to step iii) for 5 to 40 minutes.

30. A method according to any one of claims 27 to 29, wherein the biological sample is incubated for a period of 20 minutes to 16 hours