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BioAssay: AID 1622

RNAi Global Initiative pilot viability screen of human kinase and cell cycle genes

An RNAi-based silencing screen using SMARTpool siRNA silencing reagents (Thermo Fisher Scientific, Lafayette, CO, USA) targeting each of 859 human kinase and cell cycle genes that, when knocked down, induced apoptosis in HeLa cells (ATCC, CCL-2) was performed by the Luo lab at the Eppley Institute for Cancer Research, an RNAi Global Initiative member. The assay for viability was performed using more ..
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AID: 1622
Data Source: Thermo Scientific Dharmacon RNAi Technologies (luo_rgi_pilot)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
BioAssay Version:
Deposit Date: 2009-03-25
Modify Date: 2011-05-10

Data Table ( Complete ):           Active    All
Description:
An RNAi-based silencing screen using SMARTpool siRNA silencing reagents (Thermo Fisher Scientific, Lafayette, CO, USA) targeting each of 859 human kinase and cell cycle genes that, when knocked down, induced apoptosis in HeLa cells (ATCC, CCL-2) was performed by the Luo lab at the Eppley Institute for Cancer Research, an RNAi Global Initiative member. The assay for viability was performed using the CellTiter-Blue Cell Viability Assay (Promega, Madison, WI, USA), according to the manufacturer's protocol with slight modifications (See Technical Bulletin #TB317; www.promega.com/tbs/tb317/tb317.html). Genes whose knock-down was observed to either significantly induce or inhibit apoptosis were identified and are reported as active genes in this screen.
Protocol
For more details, please see a MIARE-compliant summary of the complete screen at www.rnaiglobal.org .

Cell Culture Growth Conditions: Propagation Medium was prepared by adding 100 mL FBS, 10 mL L-Glutamine and 10 mL MEM/NEAA to 880 mL of DMEM/HIGH. The total volume was 1 L (10% FBS, 2 mM L-Glutamine, and 0.1 mM NEAA). Medium was mixed thoroughly and store at 4 degrees C. Cells were propagated (4-5 days pre-transfection). 125 mL of warm (37 degrees C) Propagation Medium was measured into an appropriate container. Thawed HeLa cells were added into the Propagation Medium and mixed well. The cell suspension was distributed into (5) T175 flasks at 25 mL per flask. Flasks were incubated at 37 degrees C in a 5% CO2 incubator. Medium was replaced with fresh Propagation Medium after 16-24 hours to remove any traces of DMSO that is present in freezing medium. Incubation was continued for 5 days, at which point the plates were approximately 75-80% confluent.

Transfection: The kinase and cell cycle SMARTpool siRNA silencing reagents used in this study were designed and synthesized by Thermo Scientific, Lafayette, CO, USA. Each SMARTpool siRNA was complexed with DharmaFECT 1 transfection reagent (Thermo Fisher Scientific, Lafayette, CO, USA) for a final concentration of 50 nM per well in a 96-well plate format. Briefly, 0.75 mL DharmaFECT 1 was diluted in 74.25 mL HBSS for a total volume of diluted DharmaFECT 1 of 75 mL. Using a Multidrop Liquid Dispenser, 60.0 microL of diluted DharmaFECT 1 was dispensed into each well of 11 master screening plates in triplicate. The total volume in each well was 80.0 microL. Mixing was achieved by pipetting three times carefully up and down with a Biomek F/X liquid handling station. The mixture was incubated for 20-30 minutes at room temperature. Each target gene was evaluated by assessing cell viability using the CellTiter-Blue Cell Viability Assay according to the manufacturer's protocol with slight modifications (See Technical Bulletin #TB317; www.promega.com/tbs/tb317/tb317.html).

Delivery: To prepare cells for delivery, Propagation Medium was removed from each flask and adherent cells were washed once with 5 mL 1X PBS/flask. 2 mL of 1X trypsin was added to each flask and incubated at room temperature until cells detached from surface. Propagation Medium (8 ml) was added to each flask and the cells resuspended by gently pipetting the solution up and down. Resuspended cells from all five flasks were combined, mixed, counted, and diluted with Propagation Medium to a concentration of 1.25x105 cells/ml. Using a Biomek F/X liquid handler, 20 microL aliquots of the SMARTpool siRNA:DharmaFECT 1 complex was transferred from each master plate well into corresponding wells of each of three 96-well cell culture plates. Using a Multidrop dispenser, 80.0 microL cell suspension was added into each well. The total volume was 100 microL. The plates were incubated at 37 degrees C in 5% CO2 for 48 hours.

Viability Assay: Cell viability was assessed 48 hours after transfection with the CellTiter-Blue Cell Viability Assay, according to the manufacturer's protocol with slight modifications (See Technical Bulletin #TB317; www.promega.com/tbs/tb317/tb317.html). The 96-well cell culture plates were removed from the incubator and 5.0 microL of CellTiter-Blue reagent was added to each well. The total volume in each well was 105 microL. Plates were gently shaken and returned to the incubator for 2 hours. Plates were removed from the incubator and fluorescence measured at 544/590 nm using a FLUOstar Optima plate reader. Each 96-well assay plate took approximately 2 min for placement, file creation, and fluorescence determinations. Each plate measurement was made sequentially by plate set and order within a plate set.
Comment
This screening data was contributed by the RNAi Global Initiative. For a MIARE-compliant summary of the complete screen, please visit www.rnaiglobal.org.

HeLa cells were obtained from ATCC (CCL-2). The following were used as controls in the viability screen:
Positive controls: PLK1 SMARTpool siRNA.
Negative controls: MOCK, GAPDH SMARTpool siRNA, siCONTROL RISC-free siRNA, siCONTROL Non-Targeting siRNA pool
Active genes were identified as falling three or more standard deviations away from the sample mean. Genes falling between two and three standard deviations away from the sample mean were identified as inconclusive; all other genes were identified as inactive.

Activity scores were calculated by taking the average of the CtbZscore and the DuplicateCtbZscore (if the latter existed), multiplying by 100, and taking the absolute value of the resulting output.
Categorized Comment
Experiment Title: Pilot Screen 1

Hypothesis: To detect inter and intra lab variation in performing RNAi screens by detecting kinase and cell cycle genes that, when knocked down, induced apoptosis in HeLa cells

Sample Description: HeLa batch #1

Keywords: kinase
kinome
apoptosis
siRNA
cell cycle

Primary Contact Information: Xu Luo, Eppley Institute for Cancer Research, 987696 Nebraska Medical Center#Omaha, Nebraska 68198-7696, (T) 402-5594643, Emai: xuluo@unmc.edu

Number of distinct genes targeted for knock-down: 859

Number of replicates: 3

Description of replicates: technical replicates

Silencing RNA library description: Dharmacon

Silencing RNA reagent type: chemically synthesised siRNA - Dharmacon kinome and cell cycle library

Unique silencing RNA molecules per reagent pool: 4

Modification(s) to silencing RNA reagent
Pre-treatment
Post-treatment
Bio-material manipulations: CCI

Number of cells per well: 10000

Compound(s) name
Assay reagent name: CellTiter-Blue Cell Viability Assay

Assay reagent manufacturer: Promega

Instrument name: BMG FLUOstar Optima plate reader

Instrument manufacturer: PE

Type of readout: flourescent plate reader

Instrument settings: 0.2 s delay time, orbital reading. excitation 544 nm, emission 590 nm.

Delivery type and protocol: reverse transfection

Percentage of cell confluence: 75-80%

Complexing protocol: 0.75 mL DharmaFECT 1 was diluted in 74.25 mL HBSS. Total volume of diluted DharmaFECT 1 was 75 mL. Using a Multidrop Liquid Dispenser, 60.0 microL of diluted DharmaFECT 1 was dispensed into each well of 11 master screening plates containing siRNA. The total volume in each well was 80.0 microL. The mixture was mixed three times by pipetting (60 microl aspiration volumes) carefully up and down with a Biomek F/X liquid handling station. The mixture was incubated for 20 minutes at room temperature.##Propagation Medium was removed from each flask and adherent cells were washed once with 5 mL 1X PBS/flask. 2 mL of 1X trypsin was added to each flask and incubated at room temperature until cells detached from surface. Propagation Medium (8 ml) was added to each flask and the cells resuspended by gently pipetting the solution up and down. Resuspended cells from all five flasks were combined, mixed, counted, and diluted with Propagation Medium to a concentration of 1.25x105 cells/ml. Using a Biomek F/X liquid handler, 20 microL aliquots of the SMARTpool siRNA:DharmaFECT 1 complex was transferred from each master plate well into cooresponding wells of each of three 96-well cell culture plates. Using a Multidrop dispenser, 80.0 microL cell suspension was added into each well. The total volume was 100 microL. The plates were incubated at 37 masculineC in 5% CO2 for 48 hours.

Complexing time: 20 minutes at room temperature

Delivery reagent
Delivery reagent type: Lipid

Delivery reagent manufacturer: Dharmacon

Delivery reagent name: DharmaFECT 1

Delivery reagent final concentration: 0.15 ul/well

Silencing reagent final concentration: 50 nM

Assay plate manufacturer: Nunc, Rochester, NY, USA

Assay plate type: 96-Well Cell Culture Plates- Flat Bottom Cell Culture Plat

Media composition: DMEM/HIGH (2 x 500 mL)- Dulbecco's Modified Eagle's Medium

Time of media change
Bioactivity outcome threshold
Bioactivity score assignment method
Data normalisation method
Artefacts
Data filtering description
Data transformation details: raw fluorescence units

Analysis program: MS Excel, R, and custom C# code.

Analysis script description: MS Excel, R, and custom C# code. We used Excel and R to generate some graphs, such as histograms of controls and samples, boxplots, and replicate-to-replicate scatter plots for visual analysis. The graphs were primarily for analysis of assay quality besides using the assay quality factors, such as z-factor. We wrote C# code to calculate the scores, Z-score, robust z-score, and B-score, and assay quality factors, z-facor, z' factors and signal-to-background ratio. Our C# code also identified potential hits.

Analysis software: MS Excel, R, and custom C# code.

Quantitative data
Description of quantified data
Qualitative data
Description of qualitative data
Result Definitions
Show more
TIDNameDescriptionAnnotationHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Nucleotide ProbeIDNCBI Probe database id of reagent.Probe IDString
2GeneIDNCBI Entrez Gene database id of target gene.String
3SignalCtbRep1Signal in raw fluorescence units of Cell Titer Blue assay, replicate 1. IntegerRFU
4SignalCtbRep2Signal in raw fluorescence units of Cell Titer Blue assay, replicate 2. IntegerRFU
5SignalCtbRep3Signal in raw fluorescence units of Cell Titer Blue assay, replicate 3. IntegerRFU
6CtbZscoreZ score (number of standard deviations away from the sample mean) of the median replicate value for this reagent.Float
7DuplicateSignalCtbRep1Signal in raw fluorescence units of Cell Titer Blue assay, replicate 1 of duplicate reagent assay, where applicable.IntegerRFU
8DuplicateSignalCtbRep2Signal in raw fluorescence units of Cell Titer Blue assay, replicate 2 of duplicate reagent assay, where applicable.IntegerRFU
9DuplicateSignalCtbRep3Signal in raw fluorescence units of Cell Titer Blue assay, replicate 3 of duplicate reagent assay, where applicable.IntegerRFU
10DuplicateCtbZscoreZ score (number of standard deviations away from the sample mean) of the median replicate value for the duplicate assay of this reagent, where applicable.Float

RNAi Target.
Additional Information
Substance Type: Nucleotide

Data Table (Concise)
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