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A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Published on: July 3, 2013

Functional identification of optimized RNAi triggers using a massively parallel sensor assay.

Christof Fellmann¹, Johannes Zuber, Katherine McJunkin

¹Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.

|March 1, 2011

Summary

Developing potent short hairpin RNAs (shRNAs) for gene silencing is challenging. A new "Sensor assay" identifies effective shRNAs at scale, revealing previously unknown sequence features for optimal RNA interference.

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Area of Science:

Molecular Biology
RNA Interference (RNAi)
Gene Silencing

Background:

Short hairpin RNAs (shRNAs) are vital for gene silencing via microRNA pathways.
Inefficient shRNA design leads to poor target suppression, hindering research.
Current algorithms often miss effective shRNAs.

Purpose of the Study:

To develop a large-scale biological assay for identifying potent shRNAs.
To uncover sequence features critical for efficient shRNA biogenesis and function.
To improve the design of shRNAs for experimental applications.

Main Methods:

Creation and evaluation of 20,000 RNAi reporters across nine mammalian transcripts.
Development of a "Sensor assay" for high-throughput shRNA screening.
Unbiased analysis of sequence characteristics of effective shRNAs.

Main Results:

The "Sensor assay" reliably identifies potent shRNAs missed by existing prediction tools.
Potent shRNAs exhibit specific, previously unrecognized sequence features.
Insights into microRNA processing and strand selection were gained.

Conclusions:

The developed "Sensor assay" is a powerful tool for discovering highly effective shRNAs.
This study provides crucial insights into the sequence requirements for successful RNAi.
Improved shRNA design can enhance gene silencing experiments.