Perform genome-wide or custom boutique screens using RNAi
Post author: Kaylene Simpson. Last update: 02/11/2015 at 10:57 am by Kaylene Simpson.
The Victorian Centre for Functional Genomics at the Peter MacCallum Cancer Centre is open to the Australian and New Zealand medical research community for the purpose of providing access to siRNA, miRNA, lncRNA and shRNA knockdown screens for functional genomics studies. The Facility is managed by Dr Kaylene Simpson and overseen by a scientific advisory group consisting of scientists from Institutes across Melbourne. The VCFG has an extensive range of reagents available, principally from Dharmacon RNAi Technologies (Thermo Fisher).
siRNA: The human and mouse siRNA libraries are screened as SMARTpools in custom collections or whole genome format, followed with validation by deconvolution of the SMARTpool into individual siRNAs and screening a second SMARTpool of additional sequences termed ON-TARGETplus.
miRNA: The miRNA libraries are designed to miR Base V16 and can be screened using an overexpression (mimic) and/or knockdown (inhibitor) strategy.
lncRNA: The long non-coding RNA platform is the most recent addition to our collection, enabling siRNA-mediated knockdown of all targets currently annotated in RefSeq.
shRNA: The VCFG houses the most up to date collection of human lentiviral shRNAs in a miR30 backbone, covering 5 shRNA constructs per gene target screened in a pooled approach of thousands of constructs per pool. shRNA collections can be screened as the whole genome, boutique libraries (apoptosis, kinome, cell cylce, polarity and invasion) or user-defined collections. We have developed hpSeq amplification protocols to sequence and identify the shRNA constructs responsible for a screen phenotype and perform all our sequencing using the Molecular Genomics Facility at Peter Mac. The mouse shRNA library was purchased from Sigma (the TRC collection) and is available only in genome-wide format.
The siRNA and shRNA platforms represent complementary tools for further validation of screen hits beyond the initial identification of targets. The stable integration of shRNAs can provide long-term knockdown to follow candidates in more complex biological scenarios beyond transient 2D in vitro approaches offered by siRNA tools. Screening the protein coding genome, the miRNA and long non-coding genomes as they stand currently can now best approximate screening a true genome worth of targets.
The VCFG has a sophisticated automation platform to enable the high throughput delivery of arrayed based screens. We have developed bioinformatics pipelines for analysis of all types of screening data and have dedicated staff in the facility to assist with all elements of the screening process. The VCFG has SOPs and a detailed user guide available for all the screening stages, from assay development and optimisation, screening and analysis. We assist in conceptualising screens and writing grant applications.
The VCFG is a member of the RNAi Global Initiative (www.RNAiGlobal.org), a consortium of International Institutes that have invested in the whole genome screening technology. The VCFG represents the gene discovery arm of the Australian Phenomics Network (www.australianphenomics.org.au).
- shRNA virus production (high quality, high titre).
- automated imaging and adapting algorithms for cellular analysis.
- assay development advice.
- automated delivery of siRNA reagents and cell transfection.
Availability of technical support:
- Support for experimental design is available.
- Support for instrument use is available.
The VCFG runs a “researcher driven – staff assisted’ model where users integrate into the laboratory to perform their screens under the expertise and guidance of the VCFG. This model allows significant capacity, provides the researcher with a supportive environment in which they develop an entirely new skill set as well as generating a list of interesting new targets to follow. Interested users should contact the VCFG manager to obtain an executive summary overview of the screening activities and how to further engage with the lab.
Assay development, Bio-Assay, Bioinformatics, Bioresources, Biostatistics, Cell culture, Cells and tissues, Computational, Computational Biology, Data storage, Fluorescence, Genomics, High throughput RNAi screening, High-content systems, Light, Live-cell, Luminescence, Microscopy, Molecular libraries, Next Generation Sequencing, Quantitative image analysis, RNABiological Science categories: