Preservation of RNA in a biological sample

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.

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         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.