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Multiplex Gene Expression-Based Genome-Scale siRNA Screen to Identify Tumor-Specific Vulnerabilities in HCT116 Colon Cancer Cells - Geomean Normalized Data

PubChem AID
1259425
Source
Lewis Laboratory, University of Nebraska Medical Center
External ID
lewis_rnai_screen_geomean
BioAssay Type
Primary Screening
Substance Type
Nucleotide
Tested Substances
Version
1.1
Status
Live
Dates
  • Deposit:
    2018-08-15
  • Hold-Until:
    2019-05-25
  • Modify:
    2018-08-15
Description
Please note that the bioassay record (AID 1259425) is presented as provided to PubChem by the source(depositor). When possible, links to additional information have been provided by PubChem.

1 Description

An RNAi-based silencing screen using pooled siRNAs was used to evaluate, at genome scale, the functional similarity of each targeted gene to KSR1 to predict genes that are selectively required for survival in colon cancer cells. A high-throughput, arrayed pooled siRNA library (siGenome library - Dharmacon) targeting 14,335 genes was used to individually deplete each gene in HCT116 colon cancer cells. For each knockdown, the gene expression of eight genes was quantified using the multiplexed Affymetrix Quantigene 2.0 assay. Six of these genes (BNIP3, NDRG1, ALDOC, LOXL2, ACSL5, BNIP3L) comprised a gene expression signature that was identified based on their altered expression following KSR1 depletion. The remaining two genes (PPIB and HPRT) were included as housekeeping genes for cell number normalization. The functional similarity between individual gene depletions and KSR1 depletion was quantified based on the gene expression changes using Euclidean distance and Pearson correlation similarity metrics.

2 Protocol

HCT116 colon cancer cells were reverse transfected in 384-well plates with a final concentration of 50 nM siRNA in a transfection volume of 30 microliter containing 2,500 cells/well with 0.05 microliter Dharmafect 4 per well.

Gene expression for BNIP3L, NDRG1, ALDOC, LOXL2, BNIP3, and ACSL5, as well as control genes HPRT and PPIB was measured using branched DNA signal amplification via the Quantigene 2.0 assay (Affymetrix) based on the manufacturer's protocol.

The data has been background subtracted using "blank" wells (40 microliter of water and 30 microliter of hybridization solution) and geometric mean (geomean) normalized to correct for cell number based on the housekeeping genes (PPIB and HPRT). Some controls, including the blank wells, wells containing only Dharmafect 4 (DF4) reagents, and lethal controls (siRNA targeting PLK1 or PPIB), have been removed.

3 Comment

This PubChem submission contains the geomean-corrected (based on HPRT and PPIB reads) data for this assay. See PubChem additional PubChem submissions for the raw (lewis_rnai_screen_raw) and normalized/analyzed (lewis_rnai_screen_normalized_results) data.

PUBCHEM_ACTIVITY_OUTCOME was set to 'Unspecified' for all experimental wells and 'Inactive' for all controls including negative controls (those treated with nontargeting siRNA) and positive controls (those treated with KSR1 targeted siRNA).

4 Result Definitions

5 Data Table

7 Identity

7.1 BioAssay Name

Multiplex Gene Expression-Based Genome-Scale siRNA Screen to Identify Tumor-Specific Vulnerabilities in HCT116 Colon Cancer Cells - Geomean Normalized Data

7.2 Source

Lewis Laboratory, University of Nebraska Medical Center

7.3 External ID

lewis_rnai_screen_geomean

7.4 Project Category

RNAi Global Initiative

7.5 BioAssay Type

Primary Screening

7.6 Substance Type

Nucleotide

7.7 Deposit Date

2018-08-15

7.8 Hold-Until Date

2019-05-25

7.9 Modify Date

Version 1.1
2018-08-15 (currently shown)

7.10 Status

Live

8 Same-Project BioAssays

9 Information Sources

CONTENTS