Thorough validation of siRNA-induced cell death phenotypes defines new anti-apoptotic protein.
Loss-of-function by means of RNA interference in cultured human cells enables rapid pathway dissection on a genome-scale. Improved siRNA design and key validation protocols are required to eliminate falsely identified phenotypes resulting from potential off-target consequences. Here, we demonstrate a validation strategy involving several steps for verifying cell death phenotypes revealed during loss-of-function screening. First, from a set of 45 novel human genes we identified gene candidates that, when silenced, induce apoptosis in cultured HeLa cells. For those candidates, we performed more extensive validation with multiple effective siRNAs. In addition, we designed rescue experiments involving candidate genes delivered exogenously and containing silent mutations in the siRNA target regions. Rescue of the observed knockdown phenotype demonstrated an original and more stringent validation of the siRNA's selectivity and the phenotype specificity for the target gene. As a result, our data reveals an anti-apoptotic function for novel human breast adenocarcinoma marker BC-2, adding new depth to BC-2's description as a putative tumor marker involved in cancer related pathways.
Wu
Weilin
W
Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm, SE-17177, Sweden. weilin.wu@cgb.ki.se
Hodges
Emily
E
Höög
Christer
C
D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D004268:DNA-Binding Proteins / Q000235:genetics; D056827:Endosomal Sorting Complexes Required for Transport; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics
C429757:BTBD1 protein, human; D014408:Biomarkers, Tumor; C507674:CHMP2A protein, human; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D034741:RNA, Small Interfering; D014157:Transcription Factors; D020169:Caspases