Screening of new reference genes for qRT-PCR normalisation in rice under heat stress

qRT-PCR is one of the widely used methods for analysing the expression levels of specific genes, which requires reliable experimental results with the aid of stably-expressed reference genes. Although there are some common housekeeping genes, many experiments have proved that the expression of these genes can change under different experimental conditions. Heat stress is one of the key factors affecting rice yield and quality, and there are relatively few reports of reference genes that can be used for heat stress research. To find the reference genes that can be stably expressed under heat stress, this experiment analysed 10 common reference genes and 10 new candidate genes by qRT-PCR to explore their expression patterns in rice heat-tolerant variety HT54 and heat-sensitive variety HT13 under heat stress conditions, and used GeNorm, NormFinder, BestKeeper, ΔCT and RefFinder to evaluate the expression stability of these genes. The results showed that LOC_Os06g23160, b-TUB and eIF-4a were the most stably expressed genes in HT13 and combined analysis of these three reference genes are recommended to be used for qRT-PCR normalisation. Our results also showed that LOC_Os06g23160 was an excellent reference gene more stable than common reference genes such as 18S RNA in both HT13 and HT54.