Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies
Abstract
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(R) PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp). 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(R) gene assays. Only two of the tested Taqman(R) 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),...Continue Reading
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