Designing, optimizing, and implementing high-throughput siRNA genomic screening with glioma cells for the discovery of survival genes and novel drug targets.
A major challenge for the treatment of cancers, such as glioblastoma multiforme (GBM), has been resistance to radiation and cancer chemotherapeutics. Short interfering RNA (siRNA) based screening may facilitate the identification of genes and pathways essential for cancer cell survival and could enable a more targeted therapeutic approach for the treatment of GBM. Although the commercial availability of siRNA libraries has expanded greatly, detailed methods for the implementation and analysis of genome-scale screens are largely lacking. To annotate the essential genes and pathways for glioma cell survival, we designed, optimized, and implemented a high-throughput siRNA screen in the highly drug and radiation resistant T98G glioma cell line. We developed a rapid, readily available, and simple strategy to optimize siRNA transfection assays in a 384-well plate format based on immunofluorescence studies and inhibition of the non-essential, endogenous gene lamin A/C. We used these transfection conditions to successfully screen a library of 1056 siRNAs targeting 352 unique human genes in a cell-based one gene per well format to identify the genes essential for glioma cell survival and assess the quality of the screening conditions prior to large-scale screening. After developing and applying a median-based outlier detection algorithm for post-screen analysis, we identified the Ras oncogene family member RAN as an essential gene for glioma cell survival. Successful implementation and analysis of this siRNA screen validates our transfection optimization approach and provides guidance for the rapid development of high-throughput siRNA screens in human glioma cells.
Doris Duke Clinical Research Fellowship, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Nikhil G
NG
Thaker
Peter R
PR
McDonald
Fang
F
Zhang
Carolyn A
CA
Kitchens
Tong Ying
TY
Shun
Ian F
IF
Pollack
John S
JS
Lazo
D001331:Automation; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D055808:Drug Discovery / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods; D015698:Genomic Library; D023281:Genomics / Q000379:methods*; D005910:Glioma / Q000235:genetics; D006801:Humans; D034741:RNA, Small Interfering / Q000494:pharmacology*; D014162:Transfection / Q000379:methods
D034741:RNA, Small Interfering