Selection of reference genes in liproxstatin-1-treated K562 Leukemia cells via RT-qPCR and RNA sequencing

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) can accurately detect relative gene expression levels in biological samples. However, widely used reference genes exhibit unstable expression under certain conditions.

Here, we compared the expression stability of eight reference genes (RPLP0, RPS18, RPL13, EEF1A1, β-actin, GAPDH, HPRT1, and TUBB) commonly used in liproxstatin-1 (Lip-1)-treated K562 cells using RNA-sequencing and RT-qPCR. The expression of EEF1A1, ACTB, GAPDH, HPRT1, and TUBB was considerably lower in cells treated with 20 μM Lip-1 than in the control, and GAPDH also showed significant downregulation in the 10 μM Lip-1 group. Meanwhile, when we used geNorm, NormFinder, and BestKeeper to compare expression stability, we found that GAPDH and HPRT1 were the most unstable reference genes among all those tested. Stability analysis yielded very similar results when geNorm or BestKeeper was used but not when NormFinder was used. Specifically, geNorm and BestKeeper identified RPL13 and RPLP0 as the most stable genes under 20 μM Lip-1 treatment, whereas RPL13, EEF1A1, and TUBB were the most stable under 10 μM Lip-1 treatment. TUBB and EEF1A1 were the most stable genes in both treatment groups according to the results obtained using NormFinder. An assumed most stable gene was incorporated into each software to validate the accuracy. The results suggest that NormFinder is not an appropriate algorithm for this study.

Stable reference genes were recognized using geNorm and BestKeeper but not NormFinder. Overall, RPL13 and RPLP0 were the most stable reference genes under 20 μM Lip-1 treatment, whereas RPL13, EEF1A1, and TUBB were the most stable genes under 10 μM Lip-1 treatment.