Abstract
Background The overwhelming majority of published articles have taken colon and rectal cancer as a single group, i.e., colorectal cancer, when normalizing gene expression data with housekeeping genes (HKG) in quantitative polymerase chain reaction (qPCR) experiments though there are published reports that suggest the differential expression pattern of genes between the colon and rectal cancer groups and hence the current experiment was attempted to find out the optimal set of housekeeping genes from the list of common HKG for rectal tumor gene expression analysis. Methods The expression of five potential housekeeping genes GAPDH, RPNI, PUM1, B2M, and PMM1 was analyzed through qPCR and Bestkeeper software (http://www.wzw.tum.de/gene-quantification/bestkeeper.html) in 20 stage II-IV rectal cancer samples to check for uniformity in their expression pattern. Cancer stem cell (CSC) marker ALDH1 and epithelial-mesenchymal transition marker (EMT) markers E cadherin, vimentin, Twist, and SNAI2 expression were evaluated in conjunction with the two optimal reference genes in 10 rectal cancers as part of validation. Results The standard deviation of the cycle threshold value of GAPDH was found the lowest at 0.65 followed by RPN1 at 0.88, PUM1 at 0.94, PMM1 at 0.94, and B2M at 1.21 when analyzed with BestKeeper software. Using GAPDH and PUM1 as the reference gene for the validation phase, rectal cancer patients with stage III/IV showed a 4.79-fold change (P=0.006) in ALDH1 expression, and an 11.76-fold change in Twist expression (P=0.003) with respect to stage II rectal tumor when normalized with GAPDH and PUM1. Conclusion GAPDH and PUM1 can be used as an optimal set of housekeeping genes for gene expression-related experiments in rectal tumors. ALDH1 and Twist were found significantly overexpressed in stage III/IV rectal tumors in comparison to stage II rectal cancer. Genes associated with cancer stem cells and EMT markers could be optimally analyzed by normalizing them with GAPDH and PUM1 as housekeeping genes.