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Project 3:
Functional validationof 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”
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.
We proposeWe propose to:
SynthesizedsRNAs or ncRNA
expression constructs
Array reagentsinto screening plate
Validation andhit selection
Process cells forscreen read-out
Use establishedhigh-throughput screening assays
Data acquisition andstorage into database
Data Analysis andstatistical treatment
Meta-analysis ofresults
General experimental flow-chart
Plate cells for RNAi or
overexpression treatment
Transcriptional-LuciferaseReporter Assays
Protein modification(phospho-specific antibodies)
Microscopy-based assays
Plate reader-basedassays
GFP or antibodies
-5
0
5
10
15
20
DRSC screening platform for RNAi cell-based assays
pros: Fast, numeric data, quantitativecons: Costly, limited in information
pros: Feature rich, cheaper cons: Analysis can be challenging
(Aerius)(Discovery-1 or Opera confocal)
P
Cell number800 nm Fluorescence
P-Akt level700 nm Fluorescence
Z-scores
Multimode plate reader
Luminescence, fluorescence intensity,absorbance, FP, bidirectional stackersBarcode readerFITC, TRITC, CFP, YFP, Cy5 filter sets
Li-COR Aerius scanning reader
2 color scanning (far red) for simultaneous detectionand quantification (in-cell western)20-500 m resolutionScans microplates or membranes
Plate-reader and plate-reader/scanner
• 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
HTS microscope
Opera HTS confocal (Evotec)
DRSC Database and Website
• 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
Database
• 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
Website (http://flyrnai.org)
• 54 ncRNAs
(hand-picked by J. Manak)1
• We were able to map 45 to
unique sites
• Synthesized 1 or 2 dsRNA per
ncRNA
(72 total)
• Tested dsRNAs in the MAPK
assay2
(A. Friedman)
1 Manak et al, Nature Genet. (2006)
2 Friedman et al, Nature (2006)2
DRSC36511 targets ncRNA 2L16745000
ncRNA 2L16745000
MAPK AssayMeasure amount of dp-Erk (relative to total Erk) after EGF stimulation in S2R+ cells treated with dsRNAs for 3 days
Look NIH, we can do it…
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
miR-315 is a potent and specific activatorof Wg signaling in Drosophila
Clone 8 cells
Serena Silver, Joshua Hagen, Eric Lai
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