Experimental assessment of robust reference genes for qRT-PCR in lung cancer studies

Stable internal reference genes are crucial for quantitative real-time PCR (qRT-PCR) analyses in lung cancer studies. Widely used reference genes are mostly chosen by intuition or from pan-cancer transcriptome data and lack experimental validation by qRT-PCR in the context of lung cancer. This study evaluated the stability of candidate reference genes in lung cancer cell lines under normal homeostasis, hypoxia, and serum deprivation to screen for robust reference genes for qRT-PCR in lung cancer studies. The stability of reference gene combinations was also assessed. We found that most of the stably expressed genes from pan-cancer transcriptome analyses were not sufficiently stable under some of the tested conditions. CIAO1, CNOT4, and SNW1 were found to be the most stable reference genes under various conditions. Greater stability was achieved by combining more reference genes. We further used the hypoxia biomarker hypoxia-inducible factor (HIF)-2α to demonstrate that choosing inappropriate reference genes can lead to incorrect qRT-PCR results. We also found that the stable reference genes were irrelevant to malignancy, which may explain their stability under various conditions that cancer cells often encounter. This study provides a list of validated and stable qRT-PCR reference genes and reference gene combinations for lung cancer that may standardize qRT-PCR experiments in future lung cancer studies.