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Project 3: Functional validation of ncRNAs

Project 3: Functional validation of ncRNAs. Norbert Perrimon and Bernard Mathey-Prevot. The meaning of “Functional”. 1. “being expressed significantly above the noise”. 2. “serving a biological purpose in the context of where it is expressed”. We propose. We propose to:.

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Project 3: Functional validation of ncRNAs

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  1. Project 3: Functional validation of ncRNAs Norbert Perrimon and Bernard Mathey-Prevot

  2. The meaning of “Functional” 1. “being expressed significantly above the noise” 2. “serving a biological purpose in the context of where it is expressed” We propose We propose to: Functionally validate (through loss of function or gain of function studies) a subset of ncRNAs identified in the other Aims, using a battery of cell-based assays that we developed.

  3. Process cells for screen read-out Validation and hit selection General experimental flow-chart Synthesize dsRNAs or ncRNA expression constructs Data acquisition and storage into database Array reagents into screening plate Plate cells for RNAi or overexpression treatment Data Analysis and statistical treatment Meta-analysis of results Use established high-throughput screening assays

  4. P DRSC screening platform for RNAi cell-based assays Microscopy-based assays Plate reader-based assays Transcriptional-Luciferase Reporter Assays Protein modification (phospho-specific antibodies) GFP or antibodies (Discovery-1 or Opera confocal) (Aerius) Z-scores P-Akt level 700 nmFluorescence Cell number 800 nm Fluorescence pros: Fast, numeric data, quantitative cons: Costly, limited in information pros: Feature rich, cheaper cons: Analysis can be challenging

  5. Plate-reader and plate-reader/scanner Multimode plate reader Li-COR Aerius scanning reader Luminescence, fluorescence intensity, absorbance, FP, bidirectional stackers Barcode reader FITC, TRITC, CFP, YFP, Cy5 filter sets 2 color scanning (far red) for simultaneous detection and quantification (in-cell western) 20-500 mm resolution Scans microplates or membranes

  6. HTS microscope • Fully automated confocal imaging and autofocus system • 4 laser based excitation sources (405, 488, 532, 635 nm) • Non-confocal Epi-Fluorescence Imaging • Up to 4 independent CCD detectors • Compatible with all plate types (24 to 2080 wells) • High speed data acquisition (up to 100,000 image sets in 24 h) • On board image analysis script library and analysis • 20x (dry) and 40x, 60x water-immersion lenses Opera HTS confocal (Evotec)

  7. DRSC Database and Website Database • Internally developed LIMS suite to manage the inventory and QC of dsRNA libraries • Metadata storage • Tools and links for data normalization/analysis and for the processing of experimental data Website (http://flyrnai.org) • DRSC portal to the scientific community • • Resource for RNAi (protocols, screen data, etc.) • • Information on how to apply for a screen • • Tools and links related to RNAi and data mining

  8. Look NIH, we can do it… ncRNA 2L16745000 • 54 ncRNAs (hand-picked by J. Manak)1 • Wewere able to map 45 to unique sites • Synthesized 1 or 2 dsRNA per ncRNA (72 total) • Tested dsRNAs in the MAPK assay2 (A. Friedman) MAPK Assay Measure amount of dp-Erk (relative to total Erk) after EGF stimulation in S2R+ cells treated with dsRNAs for 3 days DRSC36511 targets ncRNA 2L16745000 1 Manak et al, Nature Genet. (2006) 2 Friedman et al, Nature (2006)2

  9. Not so fast says Sue! ncRNA 2L16745000 is in fact the 3’-UTR of CG17912 CG17912 is a confirmed hit in the MAPK screen by Friedman & Perrimon

  10. miR-315 is a potent and specific activator of Wg signaling in Drosophila Clone 8 cells Serena Silver, Joshua Hagen, Eric Lai

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