Cottonseed value can be increased by providing high-value bioactive molecules such as polyphenols for improving nutrition and health. However, there was little molecular evidence for cottonseed bioactivity in mammalian cells. Quantitative real-time-PCR (qPCR) is one widely used method for bioactivity study of natural products. It is a crucial task of qPCR data analysis using stably expressed internal reference genes. The hypothesis for reference gene selection is that a lower standard deviation of the cycle of threshold (Cq) among the treatments indicates a more stable expression of the gene. The objective of this study was to select reference genes in human colon cancer cells (COLO 205) treated with gossypol and bioactive extracts from cottonseed along with bacterial endotoxin lipopolysaccharides (LPS). SYBR Green qPCR analyzed a wide range of biomarkers at the mRNA levels involved in glucose transport, lipid biosynthesis, inflammatory response, and cancer development. 10,560 Cq values were generated from qPCR assay from 55 genes analyzed from 64 treatments with triplicate per treatment for each gene. The qPCR data showed that B-cell lymphoma 2 (Bcl2) mRNA was the most stable expressed gene among the 55 mRNAs analyzed in the human colon cancer cells. The commonly used glyceraldehyde 3 phosphate dehydrogenase (Gapdh) and ribosome protein L32 (Rpl32) mRNAs were not good qPCR references for the cells. These observations were consistent regardless of the treatment comparison between gossypol and LPS, glanded and glandless seed extracts, seed coat and kernel extracts, or treatment for 8 and 24 h. The results suggest that Bcl2 is a preferable reference gene for qPCR assays in human colon cancer cells treated with cottonseed-derived gossypol and bioactive extracts as well as LPS. The qPCR results strongly support the conclusion that the Bcl2 gene is stably expressed at the mRNA level in the human colon cancer cells regardless of the treatment, suggesting that Bcl2 gene expression is not regulated at the mRNA level but at the post-transcriptional level. These results should facilitate studies designated to evaluate bioactivity on gene expression regulation by cottonseed molecules and other natural and synthetic molecules for nutrition and health uses.

Kandan Sethumadhavan

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