Selection of microRNA expression reference genes for RT-qPCR in poplar root under low nitrogen stress

MicroRNAs (miRNAs) are regulatory RNA molecules that play a crucial role in deciphering how plants adapt to low nitrogen stress. Investigating miRNA expression levels can provide significant insights into plant responses to nitrogen deficiency. Real-time quantitative PCR (RT-qPCR) is widely employed to analyze miRNA expression under specific conditions, and the selection of appropriate reference genes is crucial for ensuring the reliability of experimental results. Nonetheless, suitable reference genes for miRNA expression studies in poplar roots under low nitrogen stress remain unidentified. This study assessed the suitability of 14 miRNAs as reference genes in poplar roots exposed to low nitrogen stress, considering their amplification efficiency and stability, as assessed using geNorm, NormFinder, deltaCt, and BestKeeper. The results indicated that miR168a and U6 snRNA emerged as the most stable reference genes under the conditions tested. Additionally, small RNA sequencing (miRNA-seq) data were employed to validate the stability of these miRNAs. The analysis demonstrated that using miR168a and U6 snRNA as reference genes yielded consistent expression profiles for miR399a, miR169c, miR171a-3p, and miR166n, aligning with the TPM values derived from miRNA-seq data. This study reported reference miRNAs in poplar roots under low nitrogen stress and provides valuable insights for advancing research on plant responses to nitrogen limitations.