Plant Breeding and Acclimatization Institute, National Research Institute, Młochów Research Center, Platanowa 19, Młochów, PL-05-831, Poland.
Department of Biology, East Carolina University, Greenville, NC, 27858, USA.
Mol Cell Probes. 2021 Feb;55:101691. doi: 10.1016/j.mcp.2020.101691. Epub 2020 Dec 25.
This was the first report on evaluating candidate reference genes for quantifying the expression profiles of both coding (e.g., mRNA) and non-coding (e.g., miRNA) genes in potato response to potato virus Y (PVY) inoculation. The reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) method was employed to quantify the expression profiles of eight selected candidate reference genes; their expression stability was analyzed by four statistical algorithms, i.e., geNorm, BestKeeper, NormFinder and RefFinder. The most stable reference genes were sEF1a, sTUBb and seIF5 with a high stability. The least stable ones were sPP2A, sSUI1 and sGAPDH. The same reference gene allows for normalization of both miRNA and mRNA levels from a single RNA sample using cDNAs synthesized in a single RT reaction, in which a stem-loop primer was used for miRNAs and the oligo (dT) for mRNAs.
这是第一篇评估候选内参基因用于定量马铃薯对马铃薯 Y 病毒 (PVY) 接种反应中编码 (如 mRNA) 和非编码 (如 miRNA) 基因表达谱的报告。采用反转录实时定量聚合酶链反应 (RT-qPCR) 方法对 8 个候选内参基因的表达谱进行定量; 采用 geNorm、BestKeeper、NormFinder 和 RefFinder 4 种统计算法分析其表达稳定性。最稳定的内参基因为 sEF1a、sTUBb 和 seIF5，稳定性较高。最不稳定的是 sPP2A、sSUI1 和 sGAPDH。同一个内参基因可以对单个 RNA 样本中的 miRNA 和 mRNA 水平进行归一化，该样本的 cDNA 是在单个 RT 反应中合成的，其中 miRNA 使用茎环引物，而 mRNA 使用 oligo (dT)。
