Evaluation of reference genes for miRNA expression analysis in Galeruca daurica (Coleoptera: Chrysomelidae) using qRT‐PCR. Issue 8 (7th June 2021)

• Record Type: Journal Article
• Authors: Wang, Hai‐Chao; Li, Ling; Li, Yan‐Yan; Tan, Yao; Pang, Bao‐Ping
• Is Part Of: Entomological research. Volume 51:Issue 8(2021)
• Page Start: 393
• Page End: 402
• Publication Date: 2021-06-07
• Subjects: expression stability -- Galeruca daurica -- microRNA -- qRT‐PCR -- reference genes; Insects -- Korea -- Periodicals; Entomology -- Periodicals; 595.709519
• DOI: 10.1111/1748-5967.12537
• Languages: English
• ISSNs: 1738-2297

Abstract

Abstract: The selection of suitable reference genes (RGs) for data normalization is a crucial step for obtaining reliable results from gene expression analysis using the quantitative real‐time polymerase chain reaction (qRT‐PCR). Expression stability of eleven putative RGs (miR‐100‐5p, miR‐92a‐3p, miR‐998‐3p, miR‐279d‐3p, miR‐305‐5p, miR‐276a‐3p, miR‐275‐3p, miR‐9a‐5p, miR‐2a‐3p, U6 snRNA and 5S rRNA) was assessed by using a web‐based tool: RefFinder, which integrates four algorithms (Delta Ct, geNorm, NormFinder, and BestKeeper), and the best combination number of RGs was calculated by geNorm. Validation of selected RGs was performed by an expression analysis of let‐7‐5p under different experimental conditions. The best combinations of RGs were shown as follows: miR‐100‐5p + miR‐275‐3p for temperature treatment, miR‐100‐5p + miR‐305‐5p for adult tissues, miR‐276a‐3p + miR‐9a‐5p for genders, miR‐2a‐3p + miR‐276a‐3p for days after eclosion, U6 snRNA + miR‐9a‐5p + miR‐2a‐3p for developmental stages, and U6 snRNA + miR‐279d‐3p for all above conditions, and there was no one RG suitable for all experimental conditions. Incorrect RG selection affected the evaluation of let‐7‐5p abundance. Our findings demonstrate that an integrated approach of multiple algorithms is necessary to identify reliable, stable RGs for miRNA evaluation, and provide a basis for further studies on expression and function analysis of miRNAs and their target genes in Galeruca daurica, a new pest with great outbreaks in Inner Mongolia since 2009.