RNA interference (RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes. The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion. In our study, a 480bp fragment of the capsid protein gene of potato virus Y (CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro, as the CP gene interferes with viral uncoating, translation and replication. A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells. CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs. Six biological replicates were performed in this study. In our findings, one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%–90%.
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Potato virus Y
fragment of capsid protein gene of Potato virus Y
small interfering RNA
Chinese hamster ovary cells
Short hairpin RNA
Reverse transcription PCR
- real-time PCR:
quantitative realtime PCR
Dulbecco’s modified eagles medium
Fetal bovine serum
- M-MuLV :
Moloney murine leukemia virus reverse transcriptase
Glyceraldehyde 3-phosphate dehydrogenase
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Tabassum, B., Nasir, I.A. & Husnain, T. Potato virus Y mRNA expression knockdown mediated by siRNAs in cultured mammalian cell line. Virol. Sin. 26, 105–113 (2011). https://doi.org/10.1007/s12250-011-3161-x
- Potato virus Y
- in-vitro Expression knockdown