RNase protection assay

Ying Jun Ma; Gregory A. Dissen; Florence Rage; Sergio R. Ojeda

doi:10.1006/meth.1996.0102

RNase protection assay

Ying Jun Ma, Gregory A. Dissen, Florence Rage, Sergio R. Ojeda

Oregon National Primate Research Center

Research output: Contribution to journal › Article › peer-review

41 Scopus citations

Abstract

The RNase protection assay is a highly sensitive technique developed to detect and measure the abundance of specific mRNAs in samples of total cellular RNA. The assay utilizes in vitro transcribed ³²P-labeled antisense RNA probes that are hybridized in solution to their complementary cellular mRNAs. This is followed by digestion of nonhybridizing (single-stranded) RNA species with RNases, removal of the RNases by treatment with proteinase K, phenol extraction of the cRNA:mRNA complexes, and electrophoretic isolation of the hybridizing cRNA fragments. Since one can synthesize 'sense' mRNAs having the same sequence as the target cellular mRNA, appropriate standard curves can be generated and used to quantitate the changes in tissue mRNA levels. Because the assay requires perfect sequence complementarity for full protection, it not only serves as a quantitative tool but also provides conclusive evidence for the existence of a specific mRNA in a given tissue. The procedure described here is a modification of that originally described by M. Gilman.

Original language	English (US)
Pages (from-to)	273-278
Number of pages	6
Journal	Methods: A Companion to Methods in Enzymology
Volume	10
Issue number	3
DOIs	https://doi.org/10.1006/meth.1996.0102
State	Published - Dec 1996

ASJC Scopus subject areas

Molecular Biology
General Biochemistry, Genetics and Molecular Biology

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10.1006/meth.1996.0102

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@article{46e4169b40c1488b9a2186b7fbf1b51b,

title = "RNase protection assay",

abstract = "The RNase protection assay is a highly sensitive technique developed to detect and measure the abundance of specific mRNAs in samples of total cellular RNA. The assay utilizes in vitro transcribed 32P-labeled antisense RNA probes that are hybridized in solution to their complementary cellular mRNAs. This is followed by digestion of nonhybridizing (single-stranded) RNA species with RNases, removal of the RNases by treatment with proteinase K, phenol extraction of the cRNA:mRNA complexes, and electrophoretic isolation of the hybridizing cRNA fragments. Since one can synthesize 'sense' mRNAs having the same sequence as the target cellular mRNA, appropriate standard curves can be generated and used to quantitate the changes in tissue mRNA levels. Because the assay requires perfect sequence complementarity for full protection, it not only serves as a quantitative tool but also provides conclusive evidence for the existence of a specific mRNA in a given tissue. The procedure described here is a modification of that originally described by M. Gilman.",

author = "Ma, {Ying Jun} and Dissen, {Gregory A.} and Florence Rage and Ojeda, {Sergio R.}",

note = "Funding Information: This work was supported by NIH Grants HD24870, HD25123, and RR00163. This is publication No. 1966 of the Oregon Regional Primate Research Center.",

year = "1996",

month = dec,

doi = "10.1006/meth.1996.0102",

language = "English (US)",

volume = "10",

pages = "273--278",

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publisher = "Academic Press Inc.",

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T1 - RNase protection assay

AU - Ma, Ying Jun

AU - Dissen, Gregory A.

AU - Rage, Florence

AU - Ojeda, Sergio R.

N1 - Funding Information: This work was supported by NIH Grants HD24870, HD25123, and RR00163. This is publication No. 1966 of the Oregon Regional Primate Research Center.

PY - 1996/12

Y1 - 1996/12

N2 - The RNase protection assay is a highly sensitive technique developed to detect and measure the abundance of specific mRNAs in samples of total cellular RNA. The assay utilizes in vitro transcribed 32P-labeled antisense RNA probes that are hybridized in solution to their complementary cellular mRNAs. This is followed by digestion of nonhybridizing (single-stranded) RNA species with RNases, removal of the RNases by treatment with proteinase K, phenol extraction of the cRNA:mRNA complexes, and electrophoretic isolation of the hybridizing cRNA fragments. Since one can synthesize 'sense' mRNAs having the same sequence as the target cellular mRNA, appropriate standard curves can be generated and used to quantitate the changes in tissue mRNA levels. Because the assay requires perfect sequence complementarity for full protection, it not only serves as a quantitative tool but also provides conclusive evidence for the existence of a specific mRNA in a given tissue. The procedure described here is a modification of that originally described by M. Gilman.

AB - The RNase protection assay is a highly sensitive technique developed to detect and measure the abundance of specific mRNAs in samples of total cellular RNA. The assay utilizes in vitro transcribed 32P-labeled antisense RNA probes that are hybridized in solution to their complementary cellular mRNAs. This is followed by digestion of nonhybridizing (single-stranded) RNA species with RNases, removal of the RNases by treatment with proteinase K, phenol extraction of the cRNA:mRNA complexes, and electrophoretic isolation of the hybridizing cRNA fragments. Since one can synthesize 'sense' mRNAs having the same sequence as the target cellular mRNA, appropriate standard curves can be generated and used to quantitate the changes in tissue mRNA levels. Because the assay requires perfect sequence complementarity for full protection, it not only serves as a quantitative tool but also provides conclusive evidence for the existence of a specific mRNA in a given tissue. The procedure described here is a modification of that originally described by M. Gilman.

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DO - 10.1006/meth.1996.0102

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JO - Methods: A Companion to Methods in Enzymology

JF - Methods: A Companion to Methods in Enzymology

IS - 3

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RNase protection assay

Abstract

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