D3038: Assessment of reference gene stability in a Diabrotica undecimpunctata cell line for quantitative real-time PCR analysis

Transcript-level analysis with quantitative real-time PCR (qRT-PCR), requires the use of reference genes for normalization. Cell lines are increasingly used as model systems for studying molecular and physiological processes since they are more easily manipulated, provide data in a shorter period of time, and can be used in high-throughput applications. The southern corn rootworm (SCR) cell line, derived from eggs of Diabrotica undecimpuctata, is an example of a model cell line that has been used for experimentation but for which little information is available about the suitability of reference genes under different experimental conditions for use in qRT-PCR analyses.  This study evaluates the stability of six reference genes: actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase, elongation factor 1 alpha, vacuolar ATPase, and ribosomal protein S3 under five experimental conditions: varying cell concentration, heat shock, treatment with a pharmacological inhibitor, exposure to double-stranded RNA, or cultured with or without fetal bovine serum. Four statistical algorithms: Delta CT, NormFinder, BestKeeper, and GeNorm-were employed to rank the stability of gene expression across these conditions using observed Ct values. Our findings indicate that different combinations of reference genes are necessary under different experimental conditions. Further, they establish a foundation for accurate transcript-level analysis using the SCR cell line in future research.