Abstract
Since the essential genes are crucial to the proliferation and survival of cancer cells, the interference of these genes is promising to be an option for cancer therapy to overcome heterogeneity. However, the essential genes are highly overestimated by RNA interference (RNAi) screenings, which is mainly caused by the pervasive off-target effect of small interference RNA (siRNA) and short hairpin RNA (shRNA). In the present study, we designed Match-Mismatch paired siRNAs to discriminate the on-target effect from off-target effect of siRNAs on cell viability. Only one of the 7 potential essential genes was validated as essential to cell viability, which demonstrates the high false positive rate in RNAi screenings. We modified the siRNA by introducing random nucleotides (N) into the guide strand to mitigate the off-target effect, without significantly compromising the on-target effect. The whole transcriptome profile analysis of cells transfected with siRNAs with or without Nindicates that siRNA-dN (with Ns on both the 2nd and the 18th bases of the guide strand) weakens the off-target effect by decreasing the unintended targets. The optimized siRNAs can be applied in the characterization of essential genes in cancer cells.
Highlights
Genome-wide sequencing has revolutionized our understanding of cancer genetics, and hundreds to thousands of mutations have been characterized in each cancer type [1,2,3,4]
Using quantitative Real-Time PCR to detect the target gene expression, we screened multiple paired Small interference RNA (siRNA) to assure that Match siRNAs had an effect on targets and Mismatch siRNAs could be used as optimal negative controls
The validation of the essential genes from the screening of human mammary cells revealed that cell viability could drop to below 50% even when the targets have no obvious depletion after the short hairpin RNA (shRNA) transfection
Summary
Genome-wide sequencing has revolutionized our understanding of cancer genetics, and hundreds to thousands of mutations have been characterized in each cancer type [1,2,3,4]. It remains difficult to determine the key mutations, which are essential to the progression of cancer cells, from hundreds of genes or functional domains alerted by somatic mutations. Essential genes are those that are indispensable for a certain organism under a certain condition [5]. High-throughput RNA interfering (RNAi) screenings are effective tools that are ubiquitously used to characterize essential genes [8, 9]. The low validation rate and the lack of overlap between different screenings demonstrate high false positive rate in RNAi screening
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