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