RNAi adds insight not only to basic biology but also gives researchers a toolkit for RNAi-based gene expression manipulation. The latest findings in the field were presented at Select Biosciences’ “RNAi Europe” conference held recently in Prague.
“One of the big concerns in this field is the specificity of RNAi compounds,” said Kristin Wiederholt, research area manager RNAi/gene regulation at Invitrogen (www.invitrogen.com). “Based on the sequence identity, these compounds can have off-target effects, inhibit unintended targets, or cause nonspecific effects.” One way to circumvent nonspecific targeting is by reducing concentrations of siRNA—but this doesn’t eliminate off-target effects.
Invitrogen’s Stealth™ RNAi, a 25-mer double-stranded RNA, is chemically modified so that only the strand that you are trying to knock down the gene of interest for works. Stealth RNAi designers also use bioinformatics to eliminate duplexes that have the potential for off-target effects by ensuring RNAi antisense reagents have a unique 2–12 region core, confirmed against the Unigene database.
Wiederholt stressed that good experimental design includes testing at least three individual nonoverlapping duplexes per target. “You want to make sure you have the same effect or phenotype or knockdown with multiple duplexes against the same target to increase your confidence level.”
As a test case, the company screened its RNAi kinase collection in a NFkB CellSensor® specific reporter based on GeneBLAzer™ reporter technology. In the primary screen, 57 targets down-regulated NFkB. Six targets were validated with a greater than 95% confidence level, including CHUK1, a known component of the NFkB pathway, and CDC2, which is involved in cell-cycle regulation.
Chemically modified duplexes, multiple duplexes, and good bioinformatic design were some of the upfront components needed for successful RNAi.