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Preservation of RNA in a biological sample

a biological sample and rna technology, applied in the field of preserving rna in biological samples, can solve the problems of limiting the information available from analysis of biological samples, and affecting the quality of biological samples,

Inactive Publication Date: 2006-07-27
KAMME FREDRIK +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the study of diseases, cell or tissue heterogeneity has limited the information available from analysis of biological samples.
Unfortunately, RNA content has been shown to be severely depleted during histochemical assays, for example, by immunostaining of tissue sections (Fink et al.
Although these protocols have had varying degrees of success, in general they have to be extremely short in duration (Fend et al.
These modified immunostaining protocols have limited usefulness because a longer incubation period is required for the better sensitivity of immunostaining detection.

Method used

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  • Preservation of RNA in a biological sample
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  • Preservation of RNA in a biological sample

Examples

Experimental program
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Effect test

example 1

RNA Diffusion Into the Tissue Incubation Buffer During Immunostaining

[0073] To characterize the mechanism by which RNA is lost from the tissue sections during immunostaining, RNA from immunostained tissue sections was extracted and compared to that from tissue sections that had not undergone immunostaining. Frozen sections were air-dried and fixed in cold acetone for 2 min. After a quick rinse in phosphate buffered saline (0.137 M NaCl, 0.0027 M KCl, 0.01 M phosphate buffer pH 7.4, PBS), sections were incubated in methyl green solution (Vector, Burlingame, Calif.) for 2 min. They were then rinsed in PBS and incubated in OX-42 antibody (Serotec, Raleigh, N.C.) diluted 1:100 in PBS with 0.5% acetylated BSA (Sigma) for 5 min. Sections were rinsed in PBS and then incubated in biotinylated goat anti-mouse antibody (Chemicon, Temecula, Calif.) diluted 1:100 in PBS with 0.5% acetylated BSA for 5 min. After rinsing in PBS, sections were incubated in 0.1 M Tris-HCl, pH 8.0 for 1 min and the...

example 2

Identification of RNA Preservatives

[0079] A testing compound, tris(4-aminophenyl)methane, was first incubated with RNA molecules in an aqueous solution, such as water. The mixture of the compound and RNA was then centrifuged at 10,000 g for 20 min to collect any precipitate. The collected precipitate was then analyzed by denaturing agarose gel electrophoresis. A band of the proper molecular weight (depending on the type of RNA used) indicated that the compound precipitated RNA.

[0080] Other compounds that precipitated RNA from an aqueous buffer included methyl green, cresyl violet, tris(4-aminophenyl)methane and hexamine cobalt. Compounds that were found positive by this screening assay were then tested for efficacy in preventing RNA loss during immunohistochemistry on brain tissue sections.

example 3

Immunohistochemical Assay

(A) Comparison Example—OX42 Immunostaining Without RNA Preservation

[0081] Frozen sections of tissues were air dried and fixed in cold acetone for 2 min. Sections were rinsed in PBS and incubated with OX42 antibody (Serotec, Raleigh, NC) diluted 1:100 in PBS with 0.5% acetylated BSA (Sigma) for 5 min. Sections were then rinsed in PBS and incubated with biotinylated goat-anti-mouse antibody (Chemicon, Temecula, Calif.), diluted 1:100 in PBS with 0.5% acetylated BSA for 5 min. Slides were again rinsed in PBS and then incubated 1 min in 100 mM Tris-HCl, pH 8. Then, slides were incubated with peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, Pa.), which was diluted 1:100 in PBS, for 5 min. Sections were rinsed in PBS, and detection was performed using AEC (DAKO, Carpinteria, Calif.) for 5 min. Sections were rinsed in water and counterstained with Mayer's Hematoxylin (BioGenex, San Ramon, Calif.) for 5 sec. Sections were rinsed...

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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.

Description

FIELD OF THE INVENTION [0001] The present invention relates to methods for preserving RNA in a biological sample undergoing analysis. More particularly, the present invention relates to methods of analyzing nucleic acid expression patterns involving the preservation of RNA in biological samples in histochemical assays. BACKGROUND OF THE INVENTION [0002] In the study of diseases, cell or tissue heterogeneity has limited the information available from analysis of biological samples. It has become increasingly important to be able to investigate mRNA expression patterns within specific cell populations at a specific physiological state. [0003] Histochemical approaches have been applied to identify specific cell populations within a biological sample. See, e.g., Okuducu et al. (2003), International Journal of Molecular Medicine 11:449-453. Such approaches include, e.g., techniques of immunohistochemistry that detect proteins, in-situ hybridization that measures messenger RNA, and fluore...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G01N1/30G01N33/48C07H21/02C07H21/04C12NC12N15/10
CPCC12N15/1003C12Q1/6841G01N1/30
Inventor KAMME, FREDRIKMEURERS, BERNHARDTALANTOV, DMITRIYU, JINGXUE
Owner KAMME FREDRIK
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