Preservation of RNA in a biological sample

• US 20040259133A1
• Filed: 04/15/2004
• Published: 12/23/2004
• Est. Priority Date: 04/25/2003
• Status: Active Grant

• Current Assignee: Bernhard H. Meurers, Dmitri Talantov, Fredrik C. Kamme, Jingxue Yu
• Original Assignee: Bernhard H. Meurers, Dmitri Talantov, Fredrik C. Kamme, Jingxue Yu
• Inventors: Talantov, Dmitri, Meurers, Bernhard H., Kamme, Fredrik C., Yu, Jingxue
• Granted Patent: US 7,056,673 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Abstract

To preserve RNA in a biological sample for analysis, the sample is incubated with an RNA preservative capable of precipitating RNA in an aqueous solution, such as a triphenylmethane dye (e.g., methyl green, crystal violet, pararosaniline, or tris-(4-aminophenyl)methane), cresyl violet, or cobalt ions. RNA preservation may be used in an immunostaining assay and other histochemical methods.

Claims

1. A method of analyzing a biological sample comprising:

preserving RNA in the biological sample by incubating the biological sample with an RNA preservative in an aqueous solution so as to precipitate RNA;

histochemically staining the RNA-preserved biological sample;

histochemically analyzing the biological sample to identify specific cell populations; and

analyzing mRNA expression patterns of the identified cells by a method comprising;

in-situ hybridization;

or

isolating identified cells and subjecting the isolated cells to bioarray gene profiling.

2. A method according to claim 1, wherein the RNA preservative is selected from the group consisting of triphenylmethane dyes, cresyl violet, polyamines, and cobalt ions.

3. A method according to claim 1, wherein the RNA preservative is a triphenylmethane dye selected from the group consisting of methyl green, crystal violet, and pararosaniline.

4. A method according to claim 1, wherein the histochemically analyzing comprises subjecting the biological sample to a histochemical assay selected from the group consisting of:

in situ hybridization for detecting mRNA;

fluorescence in-situ hybridization for detecting DNA;

immunocytochemistry assay for detecting proteins;

enzyme histochemistry assay for measuring catalytic activities of enzymes;

ligand-binding autoradiography for studying receptor-ligand interactions;

and glycohistochemistry assay for detecting carbohydrate-modified substances.

5. A method of analyzing a biological sample comprising:

(a) contacting the biological sample with an RNA-preserving solution comprising an aqueous solvent and an RNA preservative;

(b) incubating the biological sample with a buffer solution comprising an aqueous buffered solvent and a binding agent capable of binding to the biological sample;

(c) detecting the binding agent bound to the biological sample; and

(d) identifying a target cell or tissue within the biological sample based on the binding pattern of the binding agent bound to the biological sample.

6. A method according to claim 5, wherein the biological sample comprises a cell and the binding agent is a labeled molecule selected from a group consisting of:

an antibody capable of binding to an antigen of the cell;

a nucleic acid molecule capable of hybridizing to a fragment of DNA of the cell under stringent hybridization conditions;

a nucleic acid molecule capable of hybridizing to an mRNA of the cell under stringent hybridization conditions;

a lectin capable of binding to a carbohydrate-modified substance of the cell;

a substrate to an enzyme of the cell; and

a ligand capable of binding to a receptor of the cell.

7. A method according to claim 5, wherein the binding agent is a compound labeled with a radio-isotope, a fluorescent molecule, or biotin.

8. A method according to claim 5, wherein the RNA preservative is selected from the group consisting of methyl green, crystal violet, pararosaniline, tris-(4-aminophenyl)methane, cresyl violet, and hexamine cobalt.

9. A method according to claim 5, further comprising:

(e) contacting the biological sample with a labeled nucleic acid molecule capable of hybridizing to mRNA of the target cell or tissue under stringent hybridization conditions; and

(f) detecting the labeled nucleic acid molecule bound to the target cell or tissue.

10. A method according to claim 9, wherein the RNA preservative is selected from the group consisting of triphenylmethane dyes, cresyl violet, polyamines, and cobalt ions.

11. A method according to claim 5, further comprising:

(e) isolating the target cell or tissue from the biological sample;

(f) extracting mRNA from the isolated target cell or tissue; and

(g) analyzing the extracted mRNA by gene expression bioarray analysis.

12. A method according to claim 11, wherein said isolating the target cell or tissue from the biological sample comprises laser capture microdissection.

13. A method according to claim 11, further comprising amplifying the extracted mRNA from the isolated cell or tissue and labeling the amplification product.

14. A method according to claim 11, further comprising contacting the labeled amplification product with polynucleotide probes on a microarray chip under hybridization conditions sufficient to produce a hybridization pattern of complementary probe/target complexes.

15. A method according to claim 11, further comprising reverse-transcribing the extracted mRNA into cDNA.

16. A method according to claim 15, further comprising amplifying the cDNA by a multiplex polynucleotide chain reaction and labeling the amplification product.

17. A method according to claim 16, further comprising contacting the labeled amplification product with polynucleotide probes on a microarray chip under hybridization conditions sufficient to produce a hybridization pattern of complementary probe/target complexes.

18. A method according to claim 11, wherein the RNA preservative is selected from the group consisting of triphenylmethane dyes, cresyl violet, polyamines, and cobalt ions.

19. A method according to claim 11, wherein the RNA preservative is a triphenylmethane dye selected from the group consisting of methyl green, crystal violet, and pararosaniline.