Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

摘要

Background:Due to the limited RNA amounts from endomyocardial biopsies(EMBs)and low expression levels of certain genes,gene expression analyses by conventional real-time RT-PCR are restrained in EMBs.We applied two preamplification techniques,the TaqMan?PreAmp Master Mix (T-PreAmp)and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp). Results:T-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24±0.33 Ct values.The coefficients for inter-(1.89±0.48%)and intra-assay variation(0.85± 0.45%)were low for all gene assays tested(<4%).The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays(except for CD56)were-0.38± 0.33,without significant differences between self-designed and ABI inventoried Taqman?gene assays.Only two of the tested Taqman ABI inventoried gene assays(HPRT-ABI and CD56)did not maintain PreAmp uniformity levels between-1.5 and+1.5.In comparison,the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement(9.76±2.45),however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM(-3.29± 2.40;p<0.0001),and demonstrated comparable intra-assay CVs(1.47±0.74),albeit higher inter- assay CVs(5.38±2.06;p=0.01).Comparing EMBs from each 10 patients with dilated cardiomyopathy(DCM)and inflammatory cardiomyopathy(DCMi),T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27(30%)of the investigated 90 genes related to both HPRT-CCM and CDKN1B.Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.