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Open Access Highly Accessed Methodology article

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

Michel Noutsias1*, Maria Rohde1, Andrea Block1, Katrin Klippert2, Olga Lettau1, Katja Blunert13, Michael Hummel3, Uwe Kühl1, Hans Lehmkuhl4, Roland Hetzer4, Ursula Rauch1, Wolfgang Poller1, Matthias Pauschinger1, Heinz P Schultheiss1, Hans D Volk2 and Katja Kotsch2

Author Affiliations

1 Department of Cardiology and Pneumonology, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, 12200 Berlin, Germany

2 Institute of Medical Immunology, Charité – Universitätsmedizin Berlin, Campus Mitte, 10117 Berlin, Germany

3 Department of Pathology, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, 12200 Berlin, Germany

4 Deutsches Herzzentrum Berlin, 13353 Berlin, Germany

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BMC Molecular Biology 2008, 9:3  doi:10.1186/1471-2199-9-3

Published: 14 January 2008

Abstract

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.

Conclusion

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 calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.