Critical selection of internal control genes for quantitative real-time RT-PCR studies in lipopolysaccharide-stimulated human THP-1 and K562 cells

Abstract

The choice of internal control genes is important since it may affect the study outcome in RT-qPCR. Indeed, it is well-known that expression levels of traditional internal control genes can vary across tissue types and across experimental settings within one specific tissue type. The aim of this study is an evaluation of a set of housekeeping genes (HKGs) to be used in the normalization of gene expression in vitro different cultured cells, THP-1 and K562. The transcriptional stability of eleven potential internal control genes (RPL37A, ACTB, GAPDH, B2M, PPIB, PGK1, PPIA, SDHA, TBP, HPRT1 and RPL13A) were evaluated using RT-qPCR and were compared in different treatment, that was un-stimulated or LPS-stimulated cells. The raw Ct values were determined for each candidate gene at different time points following LPS-stimulated or unstimulated cells. Furthermore, all data were analyzed by the geNorm, BestKeeper, and NormFinder validation programs. Results indicated that PPIB and PGK1 were the most stable internal control genes in this study. RPL13A was found to be the least stable. This study provides the comprehensive reported assessment of internal control genes for use in expression studies in vitro cultured cells. These findings further emphasize the need to accurately validate candidate internal control genes in the study before use in gene expression studies using RT-qPCR.