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The RNAi Screening-based Target Discovery Program
Genome-Wide or Focused: All Scopes of Projects
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:
- Target Discovery screens should not focus on finding many
targets (which is usually all-too easy), but rather, on finding the best targets.
- 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;
- 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.
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Typical Project Outline
for a Genome-Scale HT-RNAi Target Discovery Screen:
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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.
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