P334Identification of suitable reference genes for gene expression studies in normal and pathological human heart tissues

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Abstract

Purpose: Quantitative real-time RT-PCR (RT-qPCR) has become the method of choice for mRNA quantification, but requires an accurate normalization based on the use of reference genes showing invariant expression across various pathological conditions. By contrast, few data exist on appropriate reference genes for human heart. Our aim was to determine a set of suitable reference genes in human atrial and ventricular tissues, from right and left cavities in control and various diseases.

Methods: Expression of 16 reference genes (ACTB, POLR2A, YWHAZ, PGK1, PPIA, GAPDH, IPO8, HMBS, GUSB, 18S, B2M, RPLP0, TBP, TFRP, UBC) was assessed in tissue from right and left ventricle from healthy and heart failure (HF) patients; tissue from right atrium from patients in sinus rhythm (SR) with or without atrium dilatation, patients with paroxysmal atrial fibrillation (AF), with chronic AF or HF; and tissue from left atrium from patients in SR and in AF. RT-qPCR was performed using Taqman® Human Endogenous Control Arrays on a 7900HT system (Applied Biosystems). Expression variability of these genes was evaluated by geNorm and Normfinder algorithms, BestKeeper software tool and comparative Delta-Ct method.

Results: Preliminary consensual analysis of the variability scores obtained for each reference gene expression shows that the most stable genes are: GUSB, IPO8, POLR2A and YWHAZ when comparing either right and left ventricle or ventricle from healthy and HF patients; POLR2A, IPO8, PPIA, HPRT1 and GAPDH when comparing either right and left atrium or atria from all pathological groups. 18S, TBP, ACTB, TFRC and B2M genes were identified as the least stable reference genes, confirming that they are not worth of selection for normalization in human heart samples.

Conclusions: The overall most stable reference genes across different heart cavities and health settings were POLR2A, IPO8, GAPDH, PPIA, YWHAZ and GUSB. RPLP0, PGK1 and HPRT1 could be also a good option for some specific experiments. This study could provide useful guidelines for reference gene selection in qRT-PCR studies in human heart.

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