RNA abundance measured by a lysate RNase protection assay
- PMID: 1381196
RNA abundance measured by a lysate RNase protection assay
Abstract
We describe a sensitive ribonuclease protection assay that we have used to measure the amount of interferon-beta RNA directly in lysates of human cells. Cell lysates were prepared in concentrated guanidine thiocyanate. Molecular hybridization with RNA probes was then performed directly in crude cell lysate, and native RNase-resistant duplexes were characterized by polyacrylamide gel electrophoresis. Comparison of interferon-beta RNA abundance by quantitative solution hybridization and lysate RNase protection showed that lysate RNase protection was highly quantitative. A high degree of reproducibility of the method was determined with a glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene probe. Sensitivity of lysate RNase protection was determined using both induced interferon-beta RNA and synthetic human endogenous reverse transcriptase RNA as _target. The lysate RNase protection method was able to measure as few as 10(4)-10(5) RNA molecules.
Similar articles
-
A lysate ribonuclease protection assay.Am Biotechnol Lab. 1994 Aug;12(9):28. Am Biotechnol Lab. 1994. PMID: 7520706 No abstract available.
-
Nonisotopic quantitation of mRNA using a novel RNase protection assay: measurement of erbB-2 mRNA in tumor cell lines.Anal Biochem. 1996 Nov 15;242(2):214-20. doi: 10.1006/abio.1996.0455. Anal Biochem. 1996. PMID: 8937564
-
Ribonuclease protection.Methods Mol Biol. 2001;181:133-44. doi: 10.1385/1-59259-211-2:133. Methods Mol Biol. 2001. PMID: 12843446
-
Multiprobe ribonuclease protection assay for simultaneous measurement of mRNA expression.Curr Protoc Immunol. 2003 Aug;Chapter 10:Unit 10.29. doi: 10.1002/0471142735.im1029s54. Curr Protoc Immunol. 2003. PMID: 18432896 Review.
-
Ribonuclease protection assay for the detection of beta 1-adrenergic receptor RNA.Methods Mol Biol. 2000;126:139-53. doi: 10.1385/1-59259-684-3:139. Methods Mol Biol. 2000. PMID: 10685407 Review. No abstract available.
Cited by
-
3'-untranslated sequences mediate post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA by 25-hydroxycholesterol.Biochem J. 1995 Apr 1;307 ( Pt 1)(Pt 1):233-8. doi: 10.1042/bj3070233. Biochem J. 1995. PMID: 7717981 Free PMC article.
-
Gradient temperature hybridization using a thermocycler for RNase protection assays.Mol Biotechnol. 2000 Jan;14(1):73-5. doi: 10.1385/MB:14:1:73. Mol Biotechnol. 2000. PMID: 10911616
-
Initiation of vesicular stomatitis virus mutant polR1 transcription internally at the N gene in vitro.J Virol. 1997 Feb;71(2):1466-75. doi: 10.1128/JVI.71.2.1466-1475.1997. J Virol. 1997. PMID: 8995672 Free PMC article.
-
Distinct mechanisms trigger apoptosis in human immunodeficiency virus type 1-infected and in uninfected bystander T lymphocytes.J Virol. 1998 Jan;72(1):660-70. doi: 10.1128/JVI.72.1.660-670.1998. J Virol. 1998. PMID: 9420271 Free PMC article.
-
Two parvoviruses that cause different diseases in mink have different transcription patterns: transcription analysis of mink enteritis virus and Aleutian mink disease parvovirus in the same cell line.J Virol. 1997 Jul;71(7):4990-6. doi: 10.1128/JVI.71.7.4990-4996.1997. J Virol. 1997. PMID: 9188563 Free PMC article.
MeSH terms
Substances
LinkOut - more resources
Other Literature Sources
Research Materials