The HT-RNAi-based Target Discovery Program

The advent of RNAi technology has finally delivered the long-sought ability to generate targeted, gene-specific loss-of-function phenotypes in the context of high throughput, genome-scale experimentation in human cells. Such phenotypes inherently offer the most direct, readily interpretable insights into gene function, while also representing the most predictive type of in vitro data available today for the development of new antagonist-type therapeutics.

The RNAi-based Target Discovery Program at Cenix has been shaped by the numerous lessons learned, the world-class tools and the fully-dedicated infrastructure carefully built-up through 11 years of extensive RNAi screening experience in several systems and disease areas. From the pioneering time-lapse microscopy screen for cell division genes in C. elegans (Gönczy et al., 2000, Nature 408: 331; Sönnichsen et al., 2005, Nature 434: 462), to studies in Drosophila cells and more recent siRNA-based screens in primary and transformed human cells, Cenix scientists have accumulated a wealth of know-how that is unparalleled elsewhere in the world (see, for example: Sachse et al., 2005. Meth. Enz. 392: 242; Sachse and Echeverri, 2004, Oncogene 23: 8384).

As a result, the HT-RNAi screening philosophy at Cenix is now guided by the following basic principles:

  1. Target Discovery screens should not focus on finding many targets (which is usually all-too easy), but rather, on finding the best targets.
  2. Since genome-scale screens invariably represent major endeavors, these should always be designed to extract a maximum depth of biological information, i.e. applying the richest possible readouts;
  3. In large scale screens, always strive towards achieving the same levels of detailed analysis typical of small scale studies;

The exact logistical design of each RNAi-based Target Discovery Project conducted at Cenix is always defined jointly with the client’s or partner’s own scientists and strategic planners. Nonetheless, the following basic multi-pass screening plan, based our experience to date, typically serves as a starting point for such studies:

  • Assay Optimization Phase: the cell-based assay(s) transferred to Cenix by the client/partner scientists must first be adapted and/or optimized to insure the best possible performance in HT-RNAi format before the screen is initiated. Experimental conditions are typically tested and/or documented here include transfection protocols, assay kinetics, selection of positive controls, sampling sizes, sample redundancy, intra-experimental variability, inter-experimental variability, etc.. If jointly-defined minimal performance criteria are not met, then the screening phase will not be initiated, and the client/partner can exit the project here.
  • Screening Phase: if assay performance is deemed adequate by both parties, the Screening Phase is initiated:
    • Pass 1: Inclusive screen. Main goal of this pass is to scale-down the problem using maximally inclusive conditions, i.e. that minimize false negatives (high rate of false positives is expected, which is easily corrected on second pass);
    • Pass 2: Specificity screen. Retain only positives that show phenotypes with multiple siRNAs;
    • Pass 3: Confirmation screen. Establish reproducibility and confirm link between observed phenotype and target down-regulation.

Throughout each project and upon its completion, all project data, from detailed protocols and raw image files to fully-processed annotations and presentation graphs, are typically delivered by Cenix to the client or partner through secure electronic formats, such that these are fully compatible with intellectual property filings and/or publications in the highest of peer-review journals.