Probing the off-target effect of EGFP siRNA and pro-siRNA in the HeLa-d1EGFP cell line

We have develped a novel method of making siRNAs (named pro-siRNA for prokaryotic siRNA). To evaluate off-targeting of pro-siRNA, we compared the mRNA expression profiles of HeLa-d1EGFP cells transfected with 4 nM EGFP siRNAs and pro-siRNAs by microarray. Overall design: We used microarray to study the off-target effect of siRNAs in the HeLa-d1EGFP cell line. After transfection of siRNAs for 24 hrs, RNA were extracted using Trizol. Deep sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #E7530). HeLa-d1EGFP cells are HeLa cells stably expressing d1EGFP gene. EGFP siRNA is a siRNA made by chemical synthesis. EGFP100 and EGFPFL are pro-siRNAs made from either a 100 bp hairpin or a full length hairpin targeting EGFP coding sequence.

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Huang L, Jin J, Deighan P, Kiner E, McReynolds L, Lieberman J