Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro

Citation
J. Winer et al., Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro, ANALYT BIOC, 270(1), 1999, pp. 41-49
Citations number
15
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ANALYTICAL BIOCHEMISTRY
ISSN journal
00032697 → ACNP
Volume
270
Issue
1
Year of publication
1999
Pages
41 - 49
Database
ISI
SICI code
0003-2697(19990515)270:1<41:DAVORQ>2.0.ZU;2-1
Abstract
In this article we present validation of a real-time RT-PCR method to quant itate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation, In real-time RT-PCR a dual-labeled fluore scent probe is degraded concomitant with PCR amplification. Input target mR NA levels are correlated with the time (measured in PCR cycles) at which th e reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultur ed cells in 96-well plates minimized DNA contamination. We show that the GA PDH gene chosen for normalization of the RNA load is truly invariant throug hout the biological treatments examined. We discuss two methods of calculat ing fold increase: a standard curve method and the Delta Delta C-t method. Real-time quantitative RT-PCR was used to determine the time course of c-fo s induction and the effect of varying doses of four known hypertrophy agent s on atrial naturitic factor messenger RNA expression in cultured cardiac m uscle cells. Our results agree with published data obtained from Northern b lot analysis. (C) 1999 Academic Press.