Quantitative PCR primer design affects quantification of dsRNA‐mediated gene knockdown

Abstract

RNA interference (RNAi) is a powerful tool for studying functions of candidate genes in both model and nonmodel organisms and a promising technique for therapeutic applications. Successful application of this technique relies on the accuracy and reliability of methods used to quantify gene knockdown. With the limitation in the availability of antibodies for detecting proteins, quantitative PCR (qPCR) remains the preferred method for quantifying target gene knockdown after dsRNA treatment. We evaluated how qPCR primer binding site and target gene expression levels affect quantification of intact mRNA transcripts following dsRNA‐mediated RNAi. The use of primer pairs targeting the mRNA sequence within the dsRNA target region failed to reveal a significant decrease in target mRNA transcripts for genes with low expression levels, but not for a highly expressed gene. By contrast, significant knockdown was detected in all cases with primer pairs targeting the mRNA sequence extending beyond the dsRNA target region, regardless of the expression levels of the target gene. Our results suggest that at least for genes with low expression levels, quantifying the efficiency of dsRNA‐mediated RNAi with primers amplifying sequences completely contained in the dsRNA target region should be avoided due to the risk of false‐negative results. Instead, primer pairs extending beyond the dsRNA target region of the mRNA transcript sequences should be used for accurate and reliable quantification of silencing efficiency.

Evaluation of non-invasive dsRNA delivery methods for the development of RNA interference in the Asian tiger mosquito Aedes albopictus

“…We showed that primers designed to hybridize within the dsRNA targeted site exhibited lower performance to quantify gene knockdown compared to primers hybridizing outside the targeted region. These ndings are consistent with previous reports [92][93][94]. This phenomenon is probably due to remnant dsRNA fragments that can accidentally be quanti ed leading to silencing underestimation.…”

Identification and characterization of soluble binding proteins associated with host foraging in the parasitoid wasp Diachasmimorpha longicaudata

“…Joint silencing was performed for OBPs 9, 10, 11, and 12 proteins, given their high similarity at the nucleotide sequence level ( S1A Fig ) and because individual silencing was not obtained in preliminary assays. However, the level of expression of each of these transcripts was analyzed by qPCR, since this technique has a higher level of accuracy at the sequence level than the RNAi, as previously described by [ 58 ]. For this purpose, two different dsRNAs (both inside the coding region: 70–469 pb) were used: ds(OBP9-12)a from 93 to 346 pb and ds(OBP9-12)b from 189 to 468 pb ( S1A Fig ).…”

Optimized protocols for RNA interference in Macrostomum lignano

“…This might be particularly true for studies of lowly-expressed genes or genes with very slowly developing phenotypes ( Hong et al . 2014 ; Onchuru and Kaltenpoth 2019 ). There are several straightforward options to sensitize experiments.…”