Selection of suitable reference genes for miRNA expression normalization by quantitative RTPCR under abiotic stress in Larix olgensis

Suitable selection of endogenous controls is necessity for obtaining reliable qRT-PCR results. However, insufficient attention has been paid to the selection of endogenous controls for normalization in different stresses in plant, especially Larix olgensis. In this study, we screened candidate internal reference genes for miRNA expression normalization after subjecting Larix olgensis PEG-induced drought, NaHCO₃, or NaCl stress. Needles samples were collected at 0, 12, 24, 48 and 96 h after treatment. Four common housekeeping genes and 11 mature miRNAs that were found to be stably expressed in small RNA sequencing data of Larix olgensis were selected as candidate internal reference genes. Their expression levels were assessed by qRT-PCR. GeNorm and NormFinder were used to analyse the stability of gene expression from the candidate internal reference genes. Finally, the expression of some miRNAs was analysed after stress. The results showed that miRNAs exhibited better expression stability compared to the analysed ncRNAs. Under PEG, NaHCO₃, or NaCl stress, miR83321, miR43169, and miR74940 were identified as the most suitable internal reference genes, respectively. Under PEG stress, the expression of most miRNAs reached its highest level at 12 h, showing a trend of first increasing expression and then decreasing. Similarly, under NaHCO₃ and NaCl stress, the expression of most miRNAs reached its maximum levels at 48 h and 12 h, respectively. This indicates that most miRNAs more quickly respond to PEG and NaCl stress than NaHCO₃ stress.