Reference gene validation for normalization of RT-qPCR assay associated with germination and survival of rice under hypoxic condition.

Study on expression of genes for the traits associated with hypoxia tolerance during the germination demands robust choice of reference genes for transcript data normalization and gene validation through real-time quantitative polymerase chain reaction (RT-qPCR). However, reliability and stability of reference genes across different rice germplasms under hypoxic condition have not been accessed yet. Stability performance of reference genes such as eukaryotic elongation factor 1 α (eEF1α), ubiquitin 10 (UBQ10), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18SrRNA), 25S ribosomal RNA (25SrRNA), β-tublin (β-TUB), actin11 (ACT11), ubiquitin C (UBC), eukaryotic elongation factor 4 α (eIF4α), and ubiquitin5 (UBQ5) was accessed through statistical algorithms like geNorm, NormFinder, Comparative ΔCt method, BestKeeper, and RefFinder in three rice germplasms (KHO, RKB, and IR-64) with varied level of tolerance to hypoxic condition during germination. Among all genes used, OsGAPDH was found to be the most suitable reference gene under hypoxic condition. The performance of the highest-ranking reference gene (OsGAPDH) in terms of stability based on statistical algorithms was further validated for its reliability and stability through RT-qPCR with hypoxia-induced target gene OsTTP7. The identified stable housekeeping gene could be used as internal control for gene expression analysis in rice under hypoxia.