FIXATION OF A BIOLOGICAL MATERIAL

Abstract

The present invention relates to a method for the treatment of a biological material, comprising the steps i) providing a biological material, and ii) contacting the biological material with a first non-aqueous composition comprising: (a1) 10 to 90 vol. % methanol, and (a2) at least one additional additive, and (a3) optionally an acid. iii) transferring the biological material into a second composition (B) comprising up to 99 vol. % ethanol as well as to a new composition for preservation of a biological material usable in said method and the biological material resulting from this method, a method for the analysis of a treated biological material, various kits as well as the use of the composition in such a method.

Claims

1.-21. (canceled)

22. A non-aqueous composition (A) for preservation of biological material, comprising (α1) 10 to less than 80 vol. % methanol, (α2) at least one additional additive, and (α3) an acid.

23. The composition according to claim 22, wherein component (α1) is present in an amount of 20% to less than 80 vol. %.

24. The composition of claim 23, wherein component (α1) is present in an amount of 30 to less than 80 vol. %.

25. The composition of claim 23, wherein compound (α1) is present in an amount of 50 to less than 80 vol. %.

26. The composition according to claim 22, wherein component (α2) is present in an amount of 1 to 50%.

27. The composition claim 22, wherein component (α3) is at least one organic acid.

28. The composition of claim 27, wherein the at least one organic acid is a weak organic acid.

29. The composition of claim 28, wherein the weak organic acid is formic acid, acetic acid, propionic acid, or a mixture thereof.

30. The composition claim 22, wherein component (α2) is selected from the group consisting of one or more detergents, one or more inhibitors that inhibit the degradation of nucleic acids or proteins, one or more viscosity regulators, one or more dyes, one or more buffer compounds, one or more preservatives, one or more complexants, one or more reducing agents, one or more substances that improve the permeability of cells, one or more chaotropic substances, one or more fixatives, one or more additional solvents that are different from methanol, and mixtures of at least two of these additives.

31. The composition according to claim 30, wherein component (α2) is selected from C.sub.2 to C.sub.12 polyols, PEG and DEGMEA.

32. The composition of claim 31, wherein the C.sub.2-C.sub.12 polyol is a diol, a thiol, or a mixture thereof.

33. The composition of claim 32, wherein the diol or thiol is 1,3-butanediol, 1,4-butanediol, 1,3-propanediol, 1,2-propanediol, 3-methyl-1,3,5-pentanetriol, 1,2,6-hexanetriol, glycerine, or glycol.

34. A kit, comprising (b1) composition comprising (α1) 10 to less than 90 vol. % methanol, (α2) at least one additional additive, and (α3) an acid, (b2) composition (B) comprising up to 99 vol. % ethanol, and (b3) optionally embedding material (C), reagents for the analysis of biomolecules in or of a biological material or for the analysis of the morphology of a biological material, or mixtures thereof.

35. A kit, comprising (b1) composition (A) comprising (α1) 10 to less than 80 vol. % methanol, (α2) at least one additional additive, and (α3) an acid, (b2) optionally composition (B) comprising up to 99 vol. % ethanol, and (b3) optionally embedding material (C), reagents for the analysis of biomolecules in or of a biological material or for the analysis of the morphology of a biological material, or mixtures thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0103] FIG. 1 shows preparations of RNA from liver tissue on a gel as explained in example 1. Lanes on the gel correspond to sample numbers of table 1.

[0104] FIG. 2 shows preparations of RNA from liver tissue on a gel as explained in example 2. Lanes on the gel correspond to sample numbers of table 2.

[0105] FIG. 3 shows preparations of RNA from liver tissue on a gel as explained in example 3. Lanes on the gel correspond to sample numbers of table 3.

[0106] FIG. 4 shows preparations of RNA from intestine tissue on a gel as explained in example 4. Lanes on the gel correspond to sample numbers of table 4.

[0107] FIG. 5 shows preparations of RNA from liver tissue on a gel as explained in example 5. Lanes on the gel correspond to sample numbers of table 5.

[0108] FIG. 6 shows preparations of RNA from liver tissue on a gel as explained in example 6. Lanes on the gel correspond to sample numbers of table 6.

[0109] FIG. 7 shows preparations of RNA from liver tissue on a gel as explained in example 7. Lanes on the gel correspond to sample numbers of table 7.

[0110] FIG. 8 shows preparations of RNA from spleen tissue on a gel as explained in example 8. Lanes on the gel correspond to sample numbers of table 8.

[0111] FIG. 9 shows preparations of RNA from spleen tissue on a gel as explained in example 9. Lanes on the gel correspond to sample numbers of table 9.

[0112] FIGS. 10A and 10B shows a fixed and stained intestine tissue, prepared according to example 10.

[0113] FIGS. 11A and 11B shows fixed and stained spleen tissue, prepared according to example 11.

[0114] FIGS. 12A and 12B shows fixed and stained spleen and kidney tissue, prepared according to example 11.

[0115] FIG. 13 shows DNA, isolated from fixed spleen tissue an a gel, prepared and isolated according to example 12.

[0116] The invention is now described in more detail by the following examples. The examples are provided for illustration only and should not be considered as limiting the invention to the shown embodiments.

Example 1

[0117] RNA Isolation from Tissue Samples Stabilised in Different Reagents According to Composition A

[0118] Liver tissue from rat was cut into pieces of approximately 5×4×4 mm directly after dissection. The samples were completely immersed into 2 to 4 ml of a fixation solution according to composition A (table 1) in a 5 ml collection vessel made of polypropylene. Tissue samples were stored for 24h at ambient temperature. RNA extraction was performed with a commercially available kit (RNeasy Mini, QIAGEN) as described in the RNeasy Mini protocol for isolation of total RNA from animal tissue. Tissue sample were cut into small pieces and placed into 2 ml microcentrifuge tubes. The weight of each piece of tissue was determined and lysis buffer (Buffer RLT, QIAGEN) containing guanidine isothiocyanate (GITC) with a volume of 350 μl per 10 mg tissue was added along with a steel ball (5 mm). Disruption and simultaneous homogenization were performed on a Mixer-Mill (Tissue-Lyser, QIAGEN) with 20 Hz for 2 min. According to the state of the art GITC lyses cells and precipitates proteins. Lysates were centrifuged using 14.000 rpm for 3 min. 350 μl supernatant, representing approximately 10 mg tissue were transferred into a new tube, mixed with 1 volume (350 μl) 70% ethanol, and applied on a silica membrane containing spin-column (RNeasy-Mini column). Lysates were transferred through the membrane by centrifugation, thereby adsorbing the RNA to the membrane. Contaminants were removed by washing the membrane twice with 350 μl GITC containing washing buffer RW1 (QIAGEN). Between the two washing steps residual DNA was removed from the membrane by pipetting 10 μl DNase (approximately 30 Kunitz units) mixed with 70 μl buffer RDD (QIAGEN) onto the membrane and incubating for 15 min at ambient temperature. After two more washing steps with 500 μl washing buffer RPE (QIAGEN), containing Tris-Cl buffer and alcohol, the membrane was dried by full speed centrifugation for 1 min at 14.000 rpm. Finally the RNA was eluted by pipetting 40 μl water followed by 1 min incubation at ambient temperature and centrifugation for 1 min at 10.000 rpm. This elution step was repeated with additional 40 μl water and both eluates were combined. All extractions were performed in triplicates.

[0119] The concentration of RNA was determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. To ensure significance, eluates were diluted with 10 mM Tris-Cl pH7.5 to show an absorbance A260 between 1 and 0.15. Under these conditions an absorbance of 1 unit at 260 nm corresponds to 44 μg RNA.

[0120] The integrity and size distribution of total RNA was analysed by denaturing agarose gel electrophoresis. For example 15 μl of eluates were mixed with 3 μl sample buffer containing formaldehyde (FA) and bromophenol blue, incubated 10 min at 70° C., chilled on ice and loaded on a 1.0% formaldehyde-agarose-MOPS gel equilibrated with 1×FA gel running buffer. Electrophoresis was performed for 90 min and approximately 3 volts per cm length of the electrophoresis chamber. RNA was visualized by ethidium bromide staining. The gel is shown as FIG. 1, lanes on the gel correspond to sample numbers shown in table 1.

[0121] As shown in table 1, treating a tissue sample with a composition according to composition A leads to high RNA yield comparable to tissue fixation with RNAlater, if tissue is stored for no longer than 24h. Shown are examples with different concentrations of component a1 and different additional components and concentrations of components a2 and a3 (table 1, 1-18 and 20-26) and RNAlater as a reference (table 1, 19).

[0122] Analysis of integrity and size distribution showed, that the ribosomal bands for 18S- and 28S rRNA appeared as sharp bands on the stained gel (FIG. 1). Since the 28S rRNA band was approximately twice as intense as the 18S RNA band, no smaller sized RNA and almost no smear were visible on the stained gel, one can conclude that the RNA sample did not suffer from degradation during fixation or preparation.

TABLE-US-00001 TABLE 1 RNA yield from 10 mg no. reagent composition tissue [μg] 1 70% Methanol, 10% glacial acetic acid, 10% PEG300, 10% triol-mixture 13.79 (25% 3-methyl-1,3,5-pentanetriol, 75% 1,2,6-hexanetriol) 10% glacial acetic acid 2 70% Methanol, 10% glacial acetic acid, 10% PEG300, 10% diethylene 15.20 glycol monoethyl ether acetate 3 70% Methanol, 10% glacial acetic acid, 10% PEG300, 10% 1,3- 19.13 butanediol 4 70% Methanol, 10% glacial acetic acid, 10% PEG300, 10% 1,4- 20.26 butanediol 5 70% Methanol, 10% glacial acetic acid, 20% PEG300 14.73 6 70% Methanol, 10% glacial acetic acid, 20% triol-mixture (25% 3- 19.17 methyl-1,3,5-pentanetriol, 75% 1,2,6-hexanetriol), 10% glacial acetic acid 7 70% Methanol, 10% glacial acetic acid, 20% diethylene glycol 29.14 monoethyl ether acetate 8 70% Methanol, 10% glacial acetic acid, 20% 1,3-butanediol 29.90 9 70% Methanol, 10% glacial acetic acid, 20% 1,4-butanediol 28.08 10 60% Methanol, 10% glacial acetic acid, 20% triol-mixture (25% 3- 17.53 methyl-1,3,5-pentanetriol, 75% 1,2,6-hexanetriol) 10% glacial acetic acid, 10% PEG300 11 60% Methanol, 10% glacial acetic acid, 20% Diethylene glycol 17.64 monoethyl ether acetate, 10% PEG300 12 60% Methanol, 10% glacial acetic acid, 20% 1,3-butanediol, 10% 18.66 PEG300 13 60% Methanol, 10% glacial acetic acid, 20% 1,4-butanediol, 10% 17.97 PEG300 14 60% Methanol, 10% glacial acetic acid, 30% PEG300 9.60 15 60% Methanol, 10% glacial acetic acid, 30% triol-mixture (25% 3- 35.03 methyl-1,3,5-pentanetriol, 75% 1,2,6-hexanetriol) 10% glacial acetic acid 16 60% Methanol, 10% glacial acetic acid, 30% diethylene glycol 28.66 monoethyl ether acetate 17 60% Methanol, 10% glacial acetic acid, 30% 1,3-butanediol 26.92 18 60% Methanol, 10% glacial acetic acid, 30% 1,4-butanediol 18.26 19 RNAlater 39.97 20 70% Methanol, 10% propionic acid, 10% PEG300, 10% LiCl (1M) 35.57 21 70% Methanol, 10% propionic acid, 10% PEG300, 10% LiCl (100 mM) 32.88 22 70% Methanol, 10% propionic acid, 10% PEG300, 10% 1,3-butanediol 40.37 23 70% Methanol, 10% propionic acid, 10% LiCl (1M), 10% 1,3-butanediol 36.19 24 70% Methanol, 10% propionic acid, 10% LiCl (100 mM), 10% 1,3- 33.14 butanediol 25 60% Methanol, 10% propionic acid, 10% PEG300, 10% LiCl (1M), 10% 29.61 1,3-butanediol 26 60% Methanol, 10% propionic acid, 10% PEG300, 10% LiCl (100 mM), 31.79 10% 1,3-butanediol

Example 2

[0123] RNA Isolation from Tissue Stabilised with Different Reagents According to Composition a as Well as Reagents Containing Ethanol as Major Component

[0124] Liver tissue from rat was cut into pieces of 5×4×4 mm directly after dissection. The samples were completely immersed into 2 to 4 ml of a fixation solution containing ethanol (table 2, 4-6) or methanol according to composition A (table 2, 1-3), in a 5 ml collection vessel made of polypropylene. Tissue samples were stored for 24h at ambient temperature.

[0125] RNA extraction and analysis of yield and integrity was performed as described in example 1. All extractions were performed in triplicates. The gel is shown as FIG. 2, lanes on the gel correspond to sample numbers shown in table 2.

[0126] As shown in table 2, RNA yield was exceptionally high when tissue samples were stabilised with reagents containing ethanol as major component. Despite the high yield, agarose gel electrophoresis showed that the RNA from ethanol containing stabilisation reagents suffered various degree of degradation during storage. In contrast to compositions 1-3 18S and/or 28S ribosomal RNA could not be stained as sharp distinct bands. Instead a smear of different degree and bands of smaller sized RNAs became visible (FIG. 2). In case of the ethanol containing reagents it can be assumed that the yield determination was artificially high due to the fact that smaller fragments of ribonucleic acids show higher absorbance at 260 nm.

TABLE-US-00002 TABLE 2 RNA yield from 10 mg no. reagent composition tissue [μg] 1 60% Methanol, 10% PEG300, 25% Diethylene 30.66 glycol monoethyl ether acetate, 5% Propionic acid 2 70% Methanol, 25% Diethylene glycol monoethyl 38.26 ether acetate, 5% Propionic acid 3 70% Methanol, 20% Diethylene glycol monoethyl 26.55 ether acetate, 10% Propionic acid 4 70% Ethanol 47.29 5 Boonfix (primary ingredient ethanol-according 50.27 to the supplier) 6 Finefix (working solution contains 70% ethanol) 52.96

Example 3

[0127] RNA Isolation from Tissue Stabilised with Different Reagents Containing Methanol as Major Component with or without Water

[0128] Liver tissue from rat was cut into pieces of approximately 5×4×4 mm directly after dissection. The samples were completely immersed into 2 to 4 ml of a fixation solution containing methanol in an aqueous solution (table 3, 1) or methanol in a non-aqueous reagent according to composition A (table 3, 2). Tissue samples were stored for an extensive time period of 4 days at ambient temperature.

[0129] RNA extraction and analysis of yield and integrity was performed as described in example 1. All extractions were performed in triplicates. The gel is shown as FIG. 3, lanes on the gel correspond to sample numbers shown in table 3.

[0130] As shown in table 3 and FIG. 3 (no. 2) even after 4 days storage intact RNA could be isolated from tissue samples stored in non-aqueous reagents according to composition A. The yield was decreased compared to the results shown in example 1 and 2, but 28S- and 18S rRNA were still visible as intact bands without smear or smaller, distinct RNA bands. In contrast the RNA from a tissue sample stabilised with an aqueous reagent was highly degraded and low concentrated (no. 1 table 3 and FIG. 3).

TABLE-US-00003 TABLE 3 RNA yield from 10 mg no. reagent composition tissue [μg] 1 60% Methanol, 30% water, 10% glacial acetic 2.25 acid 2 60% Methanol, 30% triol-mixture (25% 3- 10.98 methyl-1,3,5-pentanetriol, 75% 1,2,6-hexanetriol) 10% glacial acetic acid

Example 4

[0131] RNA Isolation from Tissue Stabilised with Different Reagents with or without Transfer into a Second Reagent with Composition B According to the Invention

[0132] Intestine tissue from rat was cut into pieces of approximately 5×4×4 mm directly after dissection. The samples were completely immersed into 5 ml of different reagents according to composition A and stored at ambient temperature. Samples were either stored for 7 days (table 4, 1-4) or transferred after 4h hours into 5 ml of a reagent according to composition B and stored within this reagent for 7 days (table 4, 5-8).

[0133] RNA extraction and analysis of yield and integrity was performed as described in example 1. All extractions were performed in triplicates. The gel is shown as FIG. 4, lanes on the gel correspond to sample numbers shown in table 4.

[0134] As shown in table 4 RNA yield dropped down significantly when samples were not transferred into the second reagent compared to those samples, which were transferred into a reagent according to composition B according to the invention. Agarose gel electrophoresis showed that even after 7 days storage intact RNA could be isolated from tissue samples stored in reagents according to composition A and B and treated according to the invention. The gel confirmed the low yield when the transfer was omitted (FIG. 4).

TABLE-US-00004 TABLE 4 RNA yield reagent from 10 mg no. reagent composition A composition B tissue [μg] 1 70% Methanol, 25% Diethylene — 3.7 glycol monoethyl ether acetate, 5% Propionic acid 2 70% Methanol, 25% 1,3- — 3.5 Butanediol, 5% Propionic acid 3 90% Methanol, 10% PEG300 — 4.4 4 70% Methanol, 30% PEG300 — 2.4 5 70% Methanol, 25% Diethylene 70% Ethanol, 30% 8.7 glycol monoethyl ether acetate, 1,3-Butanediol 5% Propionic acid 6 70% Methanol, 25% 1,3- 70% Ethanol, 30% 6.8 Butanediol, 5% Propionic acid 1,3-Butanediol 7 90% Methanol, 10% PEG300 70% Ethanol, 30% 8.7 1,3-Butanediol 8 70% Methanol, 30% PEG300 70% Ethanol, 30% 12.6 1,3-Butanediol

Example 5

[0135] RNA Isolation from Tissue Stabilised with Reagents According to Composition A and B According to the Invention

[0136] Liver tissue from rat was cut into pieces of approximately 5×4×4 mm directly after dissection. The samples were completely immersed into 10 ml of a reagent according to composition A and stored at ambient temperature. One sample was stored for 3 days (table 5, 10), the others were transferred after 2h hours into 10 ml of different reagents according to composition B and stored within these reagents for 3 days (table 5, 1-8). As a reference one sample was stored in RNAlater (table 5, 9).

[0137] RNA extraction and analysis of yield and integrity was performed as described in example 1. All extractions were performed in triplicates. The gel is shown as FIG. 5, lanes on the gel correspond to sample numbers shown in table 5

[0138] This example shows once more the effect of tissue treatment on RNA stability according to the invention, i.e. the transfer from a reagent according to composition A to a reagent according to composition B. In samples where transfer took place the RNA yield remained high in a range comparable to the reference with RNAlater (table 5, 1-7 and 9). No visible RNA degradation could be observed even after 3 days of storage (FIGS. 5, 1-7 and 9). In contrast, when the transfer was omitted, yield dropped down significantly and RNA degradation became visible by increased smear and a diminished 28S rRNA band (table 5, 10 and FIG. 5, 10). A similar effect could be observed when the sample was transferred into Boonfix (see above), which according to the supplier is a reagent consisting mainly of ethanol but is balanced with water (table 5, 8 and FIG. 5, 8).

TABLE-US-00005 TABLE 5 RNA yield from 10 mg no. reagent composition A reagent composition B tissue [μg] 1 70% Methanol, 10% Glacial acetic 70% Ethanol, 30% 1,3-Butanediol 35.90 acid/10% PEG300, 10% 1,3- Butanediol 2 70% Methanol, 10% Glacial acetic 70% Ethanol, 1% 1% Glacial acetic 33.97 acid/10% PEG300, 10% 1,3- acid, 29% 1,3-Butanediol Butanediol 3 70% Methanol, 10% Glacial acetic 70% Ethanol, 5% 1% Glacial acetic 19.93 acid/10% PEG300, 10% 1,3- acid, 25% 1,3-Butanediol Butanediol 4 70% Methanol, 10% Glacial acetic 70% Ethanol, 10% 1% Glacial 18.44 acid/10% PEG300, 10% 1,3- acetic acid, 20% 1,3-Butanediol Butanediol 5 70% Methanol, 10% Glacial acetic 60% Ethanol, 40% 1,3-Butanediol 41.68 acid/10% PEG300, 10% 1,3- Butanediol 6 70% Methanol, 10% Glacial acetic 60% Ethanol, 1% 1% Glacial acetic 33.86 acid/10% PEG300, 10% 1,3- acid, 39% 1,3-Butanediol Butanediol 7 70% Methanol, 10% Glacial acetic 60% Ethanol, 5% 1% Glacial acetic 21.02 acid/10% PEG300, 10% 1,3- acid, 35% 1,3-Butanediol Butanediol 8 70% Methanol, 10% Glacial acetic Boonfix 14.59 acid/10% PEG300, 10% 1,3- Butanediol 9 RNAlater — 37.25 10 70% Methanol, 10% Glacial acetic — 6.00 acid/10% PEG300, 10% 1,3- Butanediol

Example 6

[0139] RNA Isolation from Tissue Stabilised According to the Invention

[0140] Liver tissue from rat was cut into pieces of approximately 5×4×4 mm directly after dissection. The samples were completely immersed into 2 ml of a reagent according to a composition of step ii), stored at ambient temperature for 4 hours, transferred into different reagents according to composition B and stored for additional 2 days (table 6, 3-9). In addition one sample was not transferred but stored within the reagent composition according to step ii) (table 6, 10), one sample was transferred into 100% ethanol (table 6, 1) and one sample was transferred into 70% ethanol (table 6, 2).

[0141] RNA extraction and analysis of yield and integrity was performed as described in example 1. All extractions were performed in triplicates. The gel is shown as FIG. 6, lanes on the gel correspond to sample numbers shown in table 6.

[0142] This example demonstrates the stabilising effect according to the invention of transfer into different reagents according to composition B. Shown are different reagents with different additional components (a2) and concentrations of organic acids (a3) (table 6, 3-9 and FIG. 6, 3-9). Balancing of the second reagent with water led to decreased RNA yield and increased smear on the agarose gel, indicating advanced degradation (table and FIG. 6, 2). Omitting the transfer led to decreased RNA yield (table 6 and FIG. 6, 10). Transfer into 100% ethanol did not lead to any degradation and resulted in high RNA yield (table 6 and FIG. 6, 1). However it is commonly accepted that tissue samples stored in 100% ethanol undergo hardening and shrinkage. As a consequence the specimens become brittle which leads to artefacts when subsequent histological work methods are conducted.

TABLE-US-00006 TABLE 6 RNA yield reagent composition according from 10 mg no. to ii) reagent composition B tissue [μg] 1 60% methanol, 30% chloroform, 100% ethanol 36.51 10% glacial acetic acid 2 60% methanol, 30% chloroform, 70% ethanol, 30% 8.16 10% glacial acetic acid water 3 60% methanol, 30% chloroform, 70% ethanol, 30% 29.50 10% glacial acetic acid 1,3-ethanediol 4 60% methanol, 30% chloroform, 70% ethanol, 30% 33.22 10% glacial acetic acid diethyleneglycol mono- ethyl etheracetate 5 60% methanol, 30% chloroform, 70% ethanol, 30% 24.29 10% glacial acetic acid PEG300 6 60% methanol, 30% chloroform, 70% ethanol, 30% 29.22 10% glacial acetic acid 1,2,6-hexanetriol 7 60% methanol, 30% chloroform, 70% ethanol, 5% 9.41 10% glacial acetic acid glacial acetic acid, 25% 1,3-butanediol 8 60% methanol, 30% chloroform, 70% ethanol, 5% 16.74 10% glacial acetic acid glacial acetic acid, 25% diethyleneglycol monoethyl ether acetate 9 60% methanol, 30% chloroform, 70% ethanol, 5% 14.24 10% glacial acetic acid glacial acetic acid, 25% 1,2,6-hexanetriol 10 60% methanol, 30% chloroform, — 4.48 10% glacial acetic acid

Example 7

[0143] RNA Isolation from Tissue Stabilized According to the Invention, Replacement of Chloroform

[0144] Liver tissue from rat was cut into pieces of approximately 5×4×4 mm directly after dissection. The samples were completely immersed into 5 ml of either a chloroform containing reagent, a reagent in which chloroform is replaced by corresponding amounts of water or methanol or a reagent according to a composition of step ii), respectively, and stored at ambient temperature for 48 hours (table 7, 1-4). In addition one sample was transferred into a reagent according to composition B after 3 hours and stored for additional 45 hours (table 7, 5).

[0145] RNA extraction and analysis of yield and integrity was performed as described in example 1. All extractions were performed in triplicates. The gel is shown as FIG. 7; lanes on the gel correspond to sample numbers shown in table 7.

[0146] This example demonstrates that chloroform within a fixation reagent can not be replaced by corresponding amounts of methanol or water (table 7 and FIG. 7, 1-3). Replacing chloroform with methanol or water led to decreased RNA yields and increased smear on the agarose gel, indicating advanced degradation (table and FIGS. 7, 2 and 3). In order to achieve a similar stabilizing effect chloroform has to be replaced by at least one additional additive according to (a2) (table and FIG. 7, 4). However the best stabilizing effect with the highest RNA yield and least RNA degradation could be achieved by transferring the tissue sample after 3 hours into a reagent according to composition B (table and FIG. 7, 5).

TABLE-US-00007 TABLE 7 RNA yield reagent from 10 mg no. reagent composition composition B tissue [μg] 1 60% methanol, 30% chloroform, — 8.73 10% glacial acetic acid 2 60% methanol, 30% water, 10% — 3.04 glacial acetic acid 3 90% methanol, 10% glacial acetic — 0.96 acid 4 60% methanol, 30% — 9.42 Diethyleneglycol monoethyl ether acetate, 10% glacial acetic acid 5 60% methanol, 30% 70% Ethanol, 30% 25.13 Diethyleneglycol monoethyl ether 1,3-Butandiol acetate, 10% glacial acetic acid

Example 8

[0147] RNA Isolation from Paraffin Embedded Tissue Stabilised with Reagents According to Composition A and B According to the Invention, and Processed by a Conventional Method

[0148] Spleen tissue from rat was cut into pieces of approximately 4×4×4 mm directly after dissection. The samples were completely immersed into 5 ml of a reagent according to composition A and stored at ambient temperature for 24 hours. After this incubation period samples were either directly processed (table 8, 1 and 4) or transferred into a reagent according to composition B, stored for additional 4 days at ambient temperature (table 8, 2 and 5) or at 4° C. (table 8, 3 and 6) and finally processed.

[0149] Processing comprising of dehydration, clearing, infiltration and embedding, was performed manually following standard protocols. The specimens were placed into standard processing cassettes (histosettes) and dehydrated by transfer through increasing concentrations of ethanol, i.e. incubation in 70, 80, two times 96% ethanol, 60 min each. Clearing as the transition step between dehydration and infiltration with the embedding medium was performed by incubation twice for 60 min in 100% xylene. Tissue cavities and cells were saturated in liquid paraffin (low melting Paraplast XTRA, Roth Inc.) at 56° C. for approximately 12 hours. To provide the necessary support for microtomy specimen were embedded into the same paraffin used for infiltration.

[0150] Freshly cut sections of the paraffin blocks were used as starting material for RNA extraction. Paraffin blocks were trimmed with a rotary microtome (Leica RM2245) and 10 slices with a thickness of 10 μm each where cut off from each specimen and collected in a microcentrifuge tube. Deparaffination was performed by adding 1 ml of xylene, vortexing and centrifugation for 2 min at 14.000 rpm. The supernatant was removed and the pellet was dissolved with 1 ml of 100% ethanol. After centrifugation for 2 min at 14.000 rpm the supernatant was removed and the ethanol washing step was repeated. After centrifugation and removal of the ethanol the pellet was dissolved within 350 μl Buffer RLT (QIAGEN) containing 0.143 M ß-mercaptoethanol. For homogenisation the lysate was loaded on a QIAshredder spin column (QIAGEN) and centrifuged for 3 min at 14.000 rpm. The flowthrough was mixed with 1 volume of 70% ethanol (350 μl) and loaded on a RNeasy MinElute spin column (QIAGEN). The lysates were transferred through the membrane by centrifugation, thereby absorbing the RNA to the membrane. Washing steps with buffer RW1 and RPE as well as on membrane DNase digestion were performed as described in example 1. For elution 15 μl of water were pipetted on the silica membrane. RNA was eluted after 1 min incubation at ambient temperature by 1 min centrifugation at 14.000 rpm.

[0151] All extractions were performed in triplicates and RNA analysis of yield and integrity was performed as described in example 1. The gel is shown as FIG. 8, lanes on the gel correspond to sample numbers shown in table 8.

[0152] Analysis of RNA integrity by agarose gel electrophoresis (FIG. 8) revealed that from paraffin embedded tissue samples treated according to the invention full length RNA can be isolated. Even incubation for up to 12 hours in liquid paraffin, necessary for specimen embedding did not lead to major RNA degradation. Bands for 28S- and 18S-rRNA were still visible as sharp and distinct bands (FIG. 8). Variation of RNA yield (table 8) can be explained by different amount of starting material. In contrast to the RNA extractions from soft tissue, starting material within the slices could not be determined and normalized to e.g. 10 mg per sample. Significant differences between storage of tissue in reagent according to composition B at 4° C. or ambient temperature could not be observed.

TABLE-US-00008 TABLE 8 RNA yield from 10 slices à no. reagent composition A reagent composition B 10 μm [μg] 1 60% Methanol, 5% Propionic acid, — 3.53 10% PEG300, 25% Diethyleneglycol monoethyl ether acetate 2 60% Methanol, 5% Propionic acid, 70% Ethanol, 30% 1,3-Butanediol 1.79 10% PEG300, 25% Diethyleneglycol monoethyl ether acetate 3 60% Methanol, 5% Propionic acid, 70% Ethanol, 30% 1,3-Butanediol 1.25 10% PEG300, 25% Diethyleneglycol monoethyl ether acetate 4 70% Methanol, 10% Propionic — 2.70 acid, 20% Diethyleneglycol monoethyl ether acetate 5 70% Methanol, 10% Propionic 70% Ethanol, 30% 1,3-Butanediol 1.53 acid, 20% Diethyleneglycol monoethyl ether acetate 6 70% Methanol, 10% Propionic 70% Ethanol, 30% 1,3-Butanediol 1.39 acid, 20% Diethyleneglycol monoethyl ether acetate

Example 9

[0153] RNA Isolation from Paraffin Embedded Tissue Stabilised According to the Invention, Processed by Microwave Energy

[0154] Spleen tissue from rat was cut into pieces of approximately 4×4×4 mm directly after dissection. The samples were completely immersed into 5 ml of a reagent according to composition A. Samples were stored at ambient temperature for 24 hours (table 9, 2 and 3) or transferred after 30 min into a reagent according to composition B and stored for 24 hours at ambient temperature (table 9, 4 and 5). In parallel one sample was immersed into 5 ml of Boonfix (table 9, 1).

[0155] After 24 hours samples were placed into standard processing cassettes (histosettes) and processed on a RHS-1 microwave histoprocessor (Milestone).

[0156] For processing a four step standard protocol was applied, involving dewatering samples in 100% ethanol and heating to 65° C. by microwave energy. The ethanol was replaced by isopropanol and after air drying by vacuum and heat, specimens were infiltrated in liquid paraffin (low melting Paraplast XTRA, Roth Inc.) in a final step with heat by microwave energy and simultaneous vacuum (protocol steps see table 10). To provide the necessary support for microtomy specimen were manually embedded into the same paraffin used for infiltration.

[0157] Paraffin embedded tissue blocks were stored at ambient temperature for 5 weeks prior to RNA extraction. RNA was extracted from 10 freshly prepared slices of 10 μm thickness. Deparaffination, RNA extraction and analysis of yield and integrity were performed as described in example 8 (and 1 respectively). All extractions were performed in duplicates. The gel is shown as FIG. 9, lanes on the gel correspond to sample numbers shown in table 9.

[0158] As shown in FIG. 9, RNA from tissue samples stabilised according to the invention and processed by microwave energy was of high quality with no signs of degradation (FIG. 9, 2-5). Ribosomal bands of 28S- and 18S rRNA were sharp, with neither smear nor smaller bands detectable, in contrast to the sample treated with Boonfix (FIG. 9, 1).

TABLE-US-00009 TABLE 9 RNA yield from 10 slices no. reagent composition reagent composition B à 10 μm [μg] 1 Boonfix (primary ingredient — 2.43 ethanol-according to the supplier) 2 70% Methanol, 5% Propionic acid, 25% Diethylene glycol monoethyl — 4.54 ether acetate 3 60% Methanol, 5% Propionic acid, 25% Diethylene glycol monoethyl — 4.55 ether acetate, 10% PEG(300) 4 70% Methanol, 5% Propionic acid, 70% Ethanol, 30% 1,3-Butanediol 2.11 25% Diethylene glycol monoethyl ether acetate 5 60% Methanol, 5% Propionic acid, 70% Ethanol, 30% 1,3-Butanediol 3.55 25% Diethylene glycol monoethyl ether acetate, 10% PEG(300)

TABLE-US-00010 TABLE 10 Incubation Duration Microwave Vacuum Step medium [min] heating [mbar] Stirring 1 Ethanol 18 up to 65° C. no yes 2 Isopropanol 19 up to 68° C. no yes 3 Airdry 0.5 70° C. 500 no 4 Liquid paraffin 22 70° C. down to 100 yes

Example 10

[0159] Histological Analysis of Tissue Samples Stabilised with Reagents According to Composition A and B According to the Invention; Processed by a Conventional Method

[0160] Small intestine tissue from rat was cut into pieces with approximately 6 mm in length directly after dissection. The sample was completely immersed into 5 ml of a reagent according to composition A. After 4 hours at ambient temperature the sample was transferred into a reagent according to composition B and stored for 20 hours at ambient temperature (table 11, A). In parallel one sample was immersed into 5 ml of a 10% solution of neutral buffer formalin (NBF; table 11, B). After 24 hours samples were placed into standard processing cassettes (histosettes) and processed manually following a standard protocol starting with incubation twice in 100% ethanol for 180 min each. Clearing was performed by incubation twice for 60 min in 100% xylene. Infiltration in liquid paraffin (low melting Paraplast XTRA, Roth Inc.) was performed at 65° C. for approximately 12 hours followed by embedding into the same paraffin.

[0161] Sections of 6 μm thickness were sliced with a rotary microtome (Leica RM2245) and mounted on slides. Haematoxylin and eosin staining was performed manually with dyes from Sigma Inc., following a standard protocol (table 12).

[0162] As shown in FIGS. 10A and 10B (100-fold magnification) the section from a tissue sample stabilised according to the invention (FIG. 10A) displayed a similar morphological preservation as the section preserved in 10% neutral buffered formalin (FIG. 10B).

TABLE-US-00011 TABLE 11 no. reagent composition reagent composition B A 70% Methanol, 10% Propionic 70% Ethanol, 30% 1,3-Butanediol acid, 20% Diethylene glycol monoethyl ether acetate B NBF, 10% of neutral buffered — formalin

TABLE-US-00012 TABLE 12 Incubation/Medium Duration [min] Incubation at 70° C. 10 Rotihistol (Xylene substitute, Roth Inc.) 10 Rotihistol 10 96% Ethanol 5 80% Ethanol 5 70% Ethanol 5 60% Ethanol 5 water 3 Mayer's Haematoxylin 5 water 0.5 70% Ethanol containg 1% HCl 0.5 water 5 Eosin 5 water 1 96% Ethanol 3 96% Ethanol 5 100% Isopropanol 10 Rotihistol 10 Rotihistol 10

Example 11

[0163] Histological Analysis of Tissue Samples Stabilised with Different Reagents According to Composition a According to the Invention; Processed by a Conventional Method

[0164] Spleen and kidney from rat were cut into pieces of approximately 3×5×5 mm directly after dissection. The samples were completely immersed into 10 ml of a reagent according to composition A or in 10% neutral buffered formalin (table 13). After 24 hours at ambient temperature the samples were processed and stained. Processing of the samples was performed approximately 30 hours after dissection in a Leica TP1020 Tissue Processor. Paraffin embedded tissue samples were cut in 6 μm slices. Only the kidney sample preserved in 10% formalin was cut in 4 μm slices. Haematoxylin and eosin staining was performed on a Leica Autostainer following standard protocols identical or similar to the procedures described in detail in examples 8 and 10.

[0165] FIGS. 11A and 11B shows sections of spleen stained with haematoxylin and eosin in a 100-fold magnification. The results demonstrate similar morphological preservation between the tissue sample preserved in a reagent according to composition A (FIG. 11A) and the sample preserved in 10% neutral buffered formalin (FIG. 11B). The same result holds true for kidney, depicted in FIGS. 12A and 12B; respectively, with 200-fold magnification. The only difference observed addresses erythrocytes. In case of the fixation reagent according to composition A the erythrocytes do not contain haemoglobin.

TABLE-US-00013 TABLE 13 no. reagent composition A 70% Methanol, 10% Glacial acetic acid, 10% Diethylene glycol monoethyl ether acetate, 10% PEG300 B NBF, 10% of neutral buffered formalin

Example 12

[0166] DNA Isolation from Paraffin Embedded Tissue, Stabilised with Reagents According to Composition a According to the Invention, Processed by a Conventional Method

[0167] Spleen tissue from rat was cut into pieces of approximately 2×5×5 mm directly after dissection. The samples were completely immersed into 5 ml of different reagents according to composition A (table 14) and stored at ambient temperature for 24 hours. After this incubation period samples were manually processed as described in example 7. Paraffin embedded tissue blocks were stored at ambient temperature for 5 weeks prior to DNA extraction.

[0168] For DNA extraction, paraffin blocks were cut into half. Pure paraffin was removed and the tissue deparaffinized with xylene and ethanol (see also example 8). DNA extraction was performed with a commercially available kit (DNeasy Tissue kit, QIAGEN) as described in the DNeasy Tissue protocol for ‘purification of total DNA from animal tissues’. The pellet resulting from deparaffination was dissolved in 180 μl buffer ATL and a steel ball (5 mm) was added. Disruption and simultaneous homogenization was performed on a Mixer-Mill (Tissue-Lyser, QIAGEN) with 20 Hz for 15 seconds. The lysates were frozen at −20° C. and further processed after 24 hours by adding 40 μl proteinase K (activity 600 mAU/ml). Digestion was performed for one hour at 55° C. with constant gentle mixing of the samples. RNA was removed from the samples by adding 4 μl RNase A (100 mg/ml) and incubation for 2 min at ambient temperature. After adding 200 μl lysis buffer AL (QIAGEN), incubation for another 10 min at 70° C. and adding 200 μl ethanol (100%) the lysates were applied on a silica membrane containing DNeasy Mini spin column. Lysates were transferred through the membrane by centrifugation (1 min, 8000 rpm), thereby absorbing the DNA to the membrane. Contaminants were removed by washing the membrane with 500 μl AW1 and 500 μl AW2 (QIAGEN). After the last washing step the membrane was dried by full speed centrifugation for 3 min at 14.000 rpm. Finally the DNA was eluted by pipetting 100 μl water followed by 1 min incubation at ambient temperature and centrifugation for 1 min at 10.000 rpm. This elution step was repeated with another 100 μl water and both eluates were combined.

[0169] The concentration of DNA was determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. For DNA an absorbance of 1 unit at 260 nm corresponds to 50 μg DNA.

[0170] The integrity and size of total DNA was analysed by agarose gel electrophoresis. 400 ng DNA in 15 μl volume were mixed with 5 μl loading buffer (containing 50% glycerol and bromophenol blue). The samples were applied to 0.8% agarose gels in 1×TBE buffer. Electrophoresis was run for 120 min and approximately 3.3 Volts per cm length of the electrophoresis chamber. DNA was visualised by ethidium bromide staining.

[0171] The DNA extracted from tissue samples preserved in a reagent according to composition A, processed, embedded into paraffin blocks and stored for 5 weeks was of high molecular weight when extracted with the QIAamp DNeasy procedure. FIG. 13 shows a gel electrophoresis with 400 ng DNA from each sample (lanes correspond to sample numbers of table 14). Typical yields are listed in table 14.

TABLE-US-00014 TABLE 14 DNA no. reagent composition A yield [μg] 1 70% Methanol, 10% Propionic acid, 20% PEG300 50.10 2 70% Methanol, 10% Propionic acid, 20% triol- 41.90 mixture (25% 3-Methyl-1,3,5-pentanetriol, 75% 1,2,6-Hexanetriol) 3 70% Methanol, 10% Propionic acid, 20% 17.80 Diethylene glycol monoethyl ether acetate 4 70% Methanol, 10% Propionic acid, 20% 1,3- 33.10 Butanediol 5 70% Methanol, 10% Propionic acid, 20% 1,4- 61.30 Butanediol