Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

• Published in: BMC research notes Vol. 10; no. 1; pp. 647 - 7
• Main Authors: Mainland, Roslyn L., Lyons, Taylor A., Ruth, Mike M., Kramer, Jamie M.
• Format: Journal Article
• Language: English
• Published: London BioMed Central 29.11.2017; BioMed Central Ltd; BMC
• Subjects: Analysis; Biomedical and Life Sciences; Biomedicine; Drosophila; Drosophila transgenic RNAi lines; Genes; Genetic aspects; Genetic engineering; Genetic research; Life Sciences; Medicine/Public Health; Messenger RNA; Methods; Proteins; Quantitative real-time PCR; Research Note; RNA interference; RNAi knockdown efficiency; UAS-Gal4 system; Quantitative real-time PCR; transgenic RNAi lines; RNAi knockdown efficiency; UAS-Gal4 system; Drosophila transgenic RNAi lines
• ISSN: 1756-0500; 1756-0500
• DOI: 10.1186/s13104-017-2959-0

Objective RNA interference is employed extensively in Drosophila research to study gene function within a specific cell-type or tissue. Thousands of transgenic Drosophila lines have been generated to express double stranded RNA for gene knockdown; however, no standardized method exists for quantifying their knockdown efficiency. Since antibodies are not available for many proteins, quantitative real-time PCR is often used. Here, we explore how primer design and RNA isolation method can influence detection of gene knockdown using qPCR. Results We tested differences in detected gene knockdown efficiency when using purified polyadenylated mRNA or total RNA as templates for cDNA synthesis. We also tested two different primer locations for each gene: one to amplify a region 5′ of the RNAi cut site, and one to amplify a region 3′ of the cut site. Consistently, the strongest gene knockdown was detected when qPCR was performed using 5′ primer sets in combination with mRNA-derived cDNA. Our results indicate that detection of undegraded mRNA cleavage fragments can result in underestimation of true knockdown efficiency for a RNAi construct. Purification of polyadenylated mRNA, combined with primers designed to amplify the non-polyadenylated 5′ mRNA cleavage fragment can avoid this problem.