Title:A Simple Method for Analyzing Actives in Random RNAi Screens: Introducing the “H Score” for Hit Nomination & Gene Prioritization
VOLUME: 15 ISSUE: 9
Author(s):Bhavneet Bhinder and Hakim Djaballah*
Affiliation:HTS Core Facility, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York
Keywords:3’UTR, BDA method, esiRNA, H score, HCS, heptamer, HTS, miRNA, off-target effect, randomness, RNAi,
screening, seed sequence, shRNA, siRNA
Abstract:Due to the numerous challenges in hit identification from random RNAi screening, we have examined current
practices with a discovery of a variety of methodologies employed and published in many reports; majority of them,
unfortunately, do not address the minimum associated criteria for hit nomination, as this could potentially have been the
cause or may well be the explanation as to the lack of confirmation and follow up studies, currently facing the RNAi field.
Overall, we find that these criteria or parameters are not well defined, in most cases arbitrary in nature, and hence
rendering it extremely difficult to judge the quality of and confidence in nominated hits across published studies. For this
purpose, we have developed a simple method to score actives independent of assay readout; and provide, for the first time,
a homogenous platform enabling cross-comparison of active gene lists resulting from different RNAi screening
technologies. Here, we report on our recently developed method dedicated to RNAi data output analysis referred to as the
BDA method applicable to both arrayed and pooled RNAi technologies; wherein the concerns pertaining to inconsistent
hit nomination and off-target silencing in conjugation with minimal activity criteria to identify a high value target are
addressed. In this report, a combined hit rate per gene, called “H score”, is introduced and defined. The H score provides a
very useful tool for stringent active gene nomination, gene list comparison across multiple studies, prioritization of hits,
and evaluation of the quality of the nominated gene hits.
