Document Detail

An optimized protocol for mRNA quantification using nested competitive RT-PCR.
MedLine Citation:
PMID:  9070899     Owner:  NLM     Status:  MEDLINE    
Nested competitive reverse transcription polymerase chain reaction is an effective tool for the quantification of low level expressed mRNAs. Competitive RNA standards with small deletions allow the use cRT-PCR. The sensitivity is further increased by the utilization of nested PCR protocols. To optimize quantification of low abundance mRNAs we modified established protocols for use of automated labstations and semiautomated sequencers. In placental tissue, known for a very high CYP19 (P450AROM, aromatase) expression, cRT-PCR and nested cRT-PCR yielded virtually identical results which could be confirmed by Northern blotting. CYP19 mRNA in breast tumour tissue ranged below detection levels for Northern blotting; however, using our modified assay CYP19 showed 1.5 to 15 fold higher expression levels than in normal glandular breast tissue. Our approach proved to be useful for the quantification of a gene with low level expression. The employment of labstations and semiautomated sequencers allows rapid analysis of large sample numbers.
M Watzka; A Waha; A Koch; R K Schmutzler; F Bidlingmaier; A von Deimling; D Klingmüller; B Stoffel-Wagner
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemical and biophysical research communications     Volume:  231     ISSN:  0006-291X     ISO Abbreviation:  Biochem. Biophys. Res. Commun.     Publication Date:  1997 Feb 
Date Detail:
Created Date:  1997-04-16     Completed Date:  1997-04-16     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0372516     Medline TA:  Biochem Biophys Res Commun     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  813-7     Citation Subset:  IM    
Department of Clinical Biochemistry, University of Bonn Medical Center, Germany.
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MeSH Terms
Aromatase / genetics*
Blotting, Northern
Breast / chemistry
Gene Expression
Placenta / chemistry
Polymerase Chain Reaction / methods*
RNA, Messenger / analysis*
RNA, Neoplasm / analysis
Tissue Distribution
Reg. No./Substance:
0/RNA, Messenger; 0/RNA, Neoplasm; EC

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