Selection of best reference genes for qRT-PCR analysis of human neural stem cells preconditioned with hypoxia or baicalein-enriched fraction extracted from Oroxylum indicum medicinal plant

Whilst the potential of neural stem cell (NSC)-based treatment is recognized worldwide and seems to offer a promising therapeutic option for stroke treatments, there is currently no full understanding regarding the effects of hypoxic and baicalein-enriched fraction (BEF) preconditioning approaches on the therapeutic potential of these cells for stroke. The potential of preconditioned NSC can be determined based on the expression of several key neuroprotective genes using qRT-PCR technique. However, prior to that, it is imperative and extremely important to carefully select reference gene(s) for accurate qRT-PCR data normalization to avoid error in data interpretation. This study aimed to evaluate the stability of ten candidate reference genes via comprehensive analysis using three algorithms software: geNorm, NormFinder and BestKeeper. Our results revealed that HPRT1 and RPL13A were the most reliable reference genes for BEF-preconditioned NSCs, but ironically, HPRT1 was ranked as the least stable reference gene for hypoxic-preconditioned NSCs. On the other hand, RPLP1 and RPL13A were selected as the most stably expressed pair of reference genes for hypoxic-preconditioned NSCs. In conclusion, this study has pointed out the importance of identifying valid reference genes and has presented the first significant validation on best reference genes recommended for qRT-PCR study involves NSC preconditioned with hypoxia or with BEF extracted from Oroxylum indicum medicinal plant.