%0 Journal Article
%T Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies
%A Michel Noutsias
%A Maria Rohde
%A Andrea Block
%A Katrin Klippert
%A Olga Lettau
%A Katja Blunert
%A Michael Hummel
%A Uwe K¨¹hl
%A Hans Lehmkuhl
%A Roland Hetzer
%A Ursula Rauch
%A Wolfgang Poller
%A Matthias Pauschinger
%A Heinz P Schultheiss
%A Hans D Volk
%A Katja Kotsch
%J BMC Molecular Biology
%D 2008
%I BioMed Central
%R 10.1186/1471-2199-9-3
%X T-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 ¡À 0.33 Ct values. The coefficients for inter- (1.89 ¡À 0.48%) and intra-assay variation (0.85 ¡À 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 ¡À 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 ¡À 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 ¡À 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 ¡À 0.74), albeit higher inter-assay CVs (5.38 ¡À 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.In comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calc
%U http://www.biomedcentral.com/1471-2199/9/3