A novel highly reproducible quantitative competitve RT PCR system.
Small changes in plasma fibrinogen concentration are often clinically important. To detect changes in fibrinogen mRNA level, we have designed a novel quantitative competitive reverse transcriptase (RT) PCR system, based on our recent understanding that an important factor causing imprecision with RT PCR is a change in the initial ratio of target to standard RNA templates during reverse transcription. The system comprises: (1) titration of a fixed amount of standard RNA with four different amounts of total RNA in each tube during cDNA synthesis; (2) amplification of each of the four cDNAs with four consecutive PCR cycles; and (3) quality control reactions in which synthesised quality control RNA (rather than total RNA) is added to the standard RNA under the same RT PCR conditions as the assay reaction. To obtain reproducible data, we suggest three criteria for analysis of RT PCR results. Employing these criteria, reproducibility of RT PCR was markedly improved with an inter-assay CV of 6.5% (n=5). Using this system, we are able to detect reduction in mRNA levels of all fibrinogen genes caused by dietary protein restriction in rats after weaning (alpha 39%, p= 0. 028; beta 55%, p = 0.017; gamma 35 %, p= 0.038), with a parallel reduction of plasma fibrinogen concentration (mean (+/-SD), 157(+/-19) versus 130(+/-14) mg/dl, p=0.006).
Zhang
J
J
Department of Clinical Biochemistry , University of Cambridge, Cambridge CB2 2QR, UK.
Byrne
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CD
D000704:Analysis of Variance; D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry; D018753:Diet, Protein-Restricted; D005340:Fibrinogen / Q000032:analysis / Q000187:drug effects / Q000235:genetics; D018628:Gene Dosage; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry; D051381:Rats; D015203:Reproducibility of Results; D013698:Templates, Genetic
D018076:DNA, Complementary; D012333:RNA, Messenger; D005340:Fibrinogen; D012194:RNA-Directed DNA Polymerase