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

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