Related Experiment Video
Updated: Feb 17, 2026
Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster
Published on: August 21, 2014
Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic
Roslyn L Mainland1, Taylor A Lyons1, Mike M Ruth1
1Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, 1151 Richmond St., London, ON, N6A 5C1, Canada.
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.
