Bookmark and Share
BioAssay: AID 588793

Shh Luciferase Assay'

An RNAi-based silencing screen using On Target plus pool siRNA silencing reagents (Thermo Fisher Scientific, Lafayette, CO, USA) targeting a custom library of RFX4 regulated and other ciliary genes. This was performed by the Peterson Lab at Genentech Inc. The screen was designed to find genes involoved in receptor targeting to the cilia. Shh signaling was used as a read out for receptor targeting and readout for cilia function. The assay for viability was performed using the CellTiter-Blue Cell Viability Assay (Promega, Madison, WI, USA), according to the manufacturer's protocol. ..more
_
   
 Tested RNAi Reagents
 Tested RNAi Reagents
All(158)
 
 
Active(33)
 
 
Inactive(125)
 
 
 Screened Genes
 Screened Genes
 Related BioAssays
 Related BioAssays
Pathways (3090)
AID: 588793
Data Source: Peterson Lab, Genentech (Assay 1)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: Literature, Author
BioAssay Version:
Deposit Date: 2011-11-15
Modify Date: 2012-01-09

Data Table ( Complete ):           View Active Data    View All Data
Description:
An RNAi-based silencing screen using On Target plus pool siRNA silencing reagents (Thermo Fisher Scientific, Lafayette, CO, USA) targeting a custom library of RFX4 regulated and other ciliary genes. This was performed by the Peterson Lab at Genentech Inc. The screen was designed to find genes involoved in receptor targeting to the cilia. Shh signaling was used as a read out for receptor targeting and readout for cilia function. The assay for viability was performed using the CellTiter-Blue Cell Viability Assay (Promega, Madison, WI, USA), according to the manufacturer's protocol.
Protocol
To conduct the siRNA screen, we purchased on-target plus siRNA library targeting 160 RFX Chip-CHIP genes 4 siRNAs per gene were purchased. Dharmacon non-targeting siRNA was used as a negative control, whereas Smo siRNA and IFT88 siRNA were used as a positive control. We followed the siRNA screen protocol modified to the 384 plate format Breifly, Dharmacon libraries were aliquoted into daughter plates at a concentration of 0.5 microM, and stored at -80 degrees C. SiRNAs and Dharmafect 3 were combined to form transfection complexes in a new 96- well plate for 20 min at room temperature. The complex was aliquoted to each well and S12 cells were plated at a density of 2,200 cells per well. 3 days after transfection, 200 ng/ml Octyl-modified Shh was added to cells to induce Shh signaling. 24hr later, luciferase assay was conducted to determine Shh signaling level. Steady Lite (Perkin Elmer) was used according to the manufacturer's protocol and counts were measured with the Top Count Luminometer (ABI). The screen was done in triplicate for each treatment (with or without Shh) with a final siRNA concentration of 25 nM and both negative and positive controls were included in triplicate on each plate. Genes with more than 70% reduction of Shh signaling is considered a positive hit.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1GeneGene SymbolString
2Refseqaccession numberString
3Gene IdString
4rep 1 Z>1.5 z score replication 1Float
5rep 1 Z>2 z score replication 1Float
6rep 1 Z>3 Float
7rep 1 Z<-1.5 Float
8rep 1 Z<-2 Float
9rep 1 Z<-3 Float
10rep 2 Z>1.5 z score replication 2Float
11rep 2 Z>2 z score replication 2Float
12rep 2 Z>3 z score replication 2Float
13rep 2 Z<-1.5 z score replication 2Float
14rep 2 Z<-2 z score replication 2Float
15rep 2 Z<-3 z score replication 2Float

RNAi Target.
Additional Information
Substance Type: Nucleotide

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
PageFrom: