Identification of best housekeeping gene for normalization in qRT-PCR data under different development stages and...

The qRT-PCR has emerged as the most sought-after local gene expression profiling technique. The absolute quantification of the mRNA is highly affected by the handling artifacts. In this regard, the internal control genes serve a vital role in negating the handling errors. So far, the most stable housekeeping genes to be used for the normalization of qRT-PCR data are lacking in V. mungo, commonly known as urdbean or blackgram. Therefore, in the current study we aimed to explore the stability of the UFO, TUB2, ACT2, RHA, ISTL6, CyP, DNAJ, RBCS3B, RPS34, USP21, USB1, EfTu, UBQ10, and UBQE9 genes of V. mungo in 17 different developmental stages and 4 different abiotic stresses. The well-acknowledged algorithms such as geNorm, NormFinder, BestKeeper, the comparative ΔCt method and comprehensive RefFinder were used to assess the expression stability. According to the comprehensive ranking, RPS34 and RHA were identified as the most appropriate genes for qRT-PCR data normalization throughout all developmental stages, whereas ACT2 and RPS34 were optimal under abiotic stress conditions. Further, the suitability of these three candidate housekeeping genes for normalization has been demonstrated in an independent experiment. Based on the findings obtained, the present research advocates the use of a combination of HKGs (RPS34 and RHA for developing tissues and ACT2 and RPS34 for stress-treated tissues) for the normalization of qRT-PCR data. The proposed reference genes will guarantee the reliability and consistency of qRT-PCR results.