Reference gene validation for relative quantification analysis of transcripts in urinary exfoliated cells among urothelial bladder carcinoma patients

- Systematic validation of reference genes has been performed for RT-qPCR analysis of urinary exfoliated cells in this study.
- BestKeeper, geNorm, and NormFinder indicated similar results for the two most stable HKGs in favor of GAPDH and HSP90AB1.
- ACTB found to be the least stable one by all three algorithms.
- Among all nine studied HKGs only GAPDH, HSPCB, and UBC delivered appropriate stability, according to M values of geNorm algorithm (< 1.5).
- Using two of these HKGs would normalize target gene's expression levels more effectively based on BestKeeper algorithm.

The specific mRNA signature of urinary exfoliated cells (UECs) can be helpful for biomarker discovery and validation studies related to urological malignancies including cancers of bladder, kidney, prostate and testicles, as well as kidney allograft rejection, systemic autoimmune diseases such as lupus, and acquired proteinuric diseases. To the best of our knowledge, no systematic validation of reference genes has been reported for UECs Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) analysis up to now.To determine the stability of candidate housekeeping genes (HKGs) across UECs, they were isolated from first morning urine of patients diagnosed as high grade and low grade urothelial bladder cancer as cases and clinical controls including the patients suffering from bladder stone, benign prostatic hyperplasia, obstructive uropathy and healthy individuals. RT-qPCR was performed to determine 9 candidate HKGs stability.geNorm, Normfinder, and BestKeeper statistical algorithm were applied to determine the most stable reference genes. The non-parametric Mann-Whitney U test applied to compare HKGs expression levels between cases and controls samples.GAPDH and HSP90AB1 were selected as two most stable expressed HKGs by all three algorithms, while ACTB demonstrated to have the least stability.Although, GAPDH is a suitable reference gene for quantitative analysis of UECs in urological malignancies, based on normalization factor calculated by geNorm, we recommend using GAPDH, and HSP90AB1 together as normalizer to obtain more realistic expression analysis via relative qPCR.

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