Optimization of reference genes for qRT-PCR analysis of microRNA expression under abiotic stress conditions in sweetpotato

Sweetpotato (Ipomoea batatas. L) is an important food crop, harvested for its nutrient-rich tuberous roots. Drought and salt stresses are two major factors limiting the sweetpotato production. Since microRNAs (miRNAs) are well known to play crucial roles in regulation of plant stress responses, quantitative profiling of miRNA expression under stress conditions will facilitate identification and genetic manipulation of novel miRNAs to improve stress tolerance. Real-time quantitative reverse transcription PCR (qRT-PCR) is a commonly used tool for this purpose, but not without challenges. Although stem-loop and poly(A)-tail modified qRT-PCR methods were developed for characterizing miRNA expression, accurate profiling of miRNAs is still difficult in many plant species because of a lack of reliable reference genes for normalizing miRNA transcripts. To identify reference genes that are suitable for normalizing miRNA expression in sweetpotato, the expression stability of eight candidate miRNAs and two commonly used reference genes were tested in 96 samples involving four tissues and two cultivars under drought and salt stress treatments. Data analysis using the geNorm, NormFinder and Bestkeeper algorithms demonstrated that miRn60, miR482, and their combination were reliable references. We further validated the reference genes by expression analysis of the well-characterized miR319 and miR156 that regulate drought and salt stress responses, respectively. The reference genes identified in this study will facilitate future miRNA analysis under abiotic stress conditions in sweetpotato.