Effect of RNA integrity on reference gene expression stability in human post-mortem wounded and normal skin

Abstract

Wound age estimation has focused on characteristic time-dependent gene expression during the wound healing process according to animal models. Evaluation of these findings with relative qPCR requires cautious normalisation, particularly for human post-mortem tissues. In this study, total RNA was extracted from human post-mortem wounded and reference (non-injured) skin tissue. The obtained RNA Integrity Numbers (RINs) ranged from 2 to 7.8 (mean 4.4±1.8). RNA samples were subdivided into two subgroups within the reference and wounded groups according to the extent of degradation (moderate RIN≥4 and≤7.8; extensive RIN<4). Both moderately and extensively degraded groups were tested by analysing the expression of four endogenous control genes: GAPDH, PGK1, PPIA and YWHAZ. Notably, the subdivision based on RNA integrity affected the gene expression stabilities (M values) assessed by the reference gene validation software geNorm. In the moderately degraded reference skin, all four genes yielded high expression stability (M<1). Conversely, the extensively degraded reference samples showed reduced expression stability (M>1) and altered gene order. For wounded tissue, extended RNA degradation resulted only in reduced stability (higher M values) of all reference genes, without changes in gene stability order. GAPDH and PGK1 remained as the two most stable genes across all degradation degrees and tissue types. In total, higher RNA integrity resulted in higher reference gene expression stability and vice-versa. The results of this study highlight the importance of RNA integrity research when addressing the normalisation of target genes involved in the molecular response to wound healing.