Identification of optimal reference genes for transcriptomic analyses in normal and diseased human heart

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. Only few data exist on appropriate reference genes for the human heart. The objective of this study was to determine a set of suitable reference genes in human atrial and ventricular tissues, from right and left cavities in control and in cardiac diseases.

We assessed the expression of 16 reference genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC, YWHAZ, 18S) in tissues from: right and left ventricles from healthy controls and heart failure (HF) patients; right-atrial tissue from patients in sinus rhythm with (SRd) or without (SRnd) atrial dilatation, patients with paroxysmal (pAF) or chronic (cAF) atrial fibrillation or with HF; and left-atrial tissue from patients in SR or cAF. Consensual analysis (by geNorm and Normfinder algorithms, BestKeeper software tool and comparative delta-Ct method) of the variability scores obtained for each reference gene expression shows that the most stably expressed genes are: GAPDH, GUSB, IPO8, POLR2A, and YWHAZ when comparing either right and left ventricle or ventricle from healthy controls and HF patients; GAPDH, IPO8, POLR2A, PPIA, and RPLP0 when comparing either right and left atrium or right atria from all pathological groups. ACTB, TBP, TFRC, and 18S genes were identified as the least stable.

The overall most stable reference genes across different heart cavities and disease conditions were GAPDH, IPO8, POLR2A and PPIA. YWHAZ or GUSB could be added to this set for some specific experiments. This study should provide useful guidelines for reference gene selection in RT-qPCR studies in human heart.