tetano
Editor, Senior Moderator
J King Saud Univ Sci
. 2023 Jan 5;102540.
doi: 10.1016/j.jksus.2023.102540. Online ahead of print.
Delivery of siRNAs against MERS-CoV in Vero and HEK-293 cells: A comparative evaluation of transfection reagents
Sayed Sartaj Sohrab[SUP] 1 2 [/SUP], Sherif Aly El-Kafrawy[SUP] 1 2 [/SUP], Zeenat Mirza[SUP] 2 3 [/SUP], Ahmed M Hassan[SUP] 1 [/SUP], Fatima Alsaqaf[SUP] 1 [/SUP], Esam Ibraheem Azhar[SUP] 1 2 [/SUP]
Affiliations
Abstract
Background: A new coronavirus was identified in Jeddah, Saudi Arabia in 2012 and designated as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). To date, this virus has been reported in 27 countries. The virus transmission to humans has already been reported from camels. Currently, there is no vaccine or antiviral therapy available against this virus.
Methods: The siRNAs were in silico predicted, designed, and chemically synthesized by using the MERS-CoV-orf1ab region as a target. The antiviral activity was experimentally evaluated by delivering the siRNAs with Lipofectamine™ 2000 and JetPRIME[SUP]R[/SUP] as transfection reagents in both Vero cell and HEK-293-T cell lines at two different concentrations (10.0nM and 5.0 nM). The Ct value of quantitative Real-Time PCR (qRT-PCR) was used to calculate and determine the reduction of viral RNA level in both cell supernatant and cell lysate isolated from both cell lines.
Results: The sequence alignment resulted in the selection of highly conserved regions. The orf1ab region was used to predict and design the siRNAs and a total of twenty-one siRNAs were finally selected from four hundred and twenty-six siRNAs generated by online software. Inhibition of viral replication and significant reduction of viral RNA was observed against selected siRNAs in both cell lines at both concentrations. Based on the Ct value, the siRNAs # 11, 12, 18, and 20 were observed to be the best performing in both cell lines at both concentrations.
Conclusion: Based on the results and data analysis, it is concluded that the use of two different transfection reagents was significantly effective. But the Lipofectamine™ 2000 was found to be a better transfection reagent than the JetPRIME[SUP]R[/SUP] for the delivery of siRNAs in both cell lines.
Keywords: HEK-293-T cells; In silico prediction; MERS-CoV; MERS-CoV, Middle East respiratory Syndrome Coronavirus; Saudi Arabia; Vero Cells; qRT-PCR, quantitative Real-Time PCR; siRNAs; siRNAs, short interfering RNAs.
. 2023 Jan 5;102540.
doi: 10.1016/j.jksus.2023.102540. Online ahead of print.
Delivery of siRNAs against MERS-CoV in Vero and HEK-293 cells: A comparative evaluation of transfection reagents
Sayed Sartaj Sohrab[SUP] 1 2 [/SUP], Sherif Aly El-Kafrawy[SUP] 1 2 [/SUP], Zeenat Mirza[SUP] 2 3 [/SUP], Ahmed M Hassan[SUP] 1 [/SUP], Fatima Alsaqaf[SUP] 1 [/SUP], Esam Ibraheem Azhar[SUP] 1 2 [/SUP]
Affiliations
- PMID: 36624781
- PMCID: PMC9814285
- DOI: 10.1016/j.jksus.2023.102540
Abstract
Background: A new coronavirus was identified in Jeddah, Saudi Arabia in 2012 and designated as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). To date, this virus has been reported in 27 countries. The virus transmission to humans has already been reported from camels. Currently, there is no vaccine or antiviral therapy available against this virus.
Methods: The siRNAs were in silico predicted, designed, and chemically synthesized by using the MERS-CoV-orf1ab region as a target. The antiviral activity was experimentally evaluated by delivering the siRNAs with Lipofectamine™ 2000 and JetPRIME[SUP]R[/SUP] as transfection reagents in both Vero cell and HEK-293-T cell lines at two different concentrations (10.0nM and 5.0 nM). The Ct value of quantitative Real-Time PCR (qRT-PCR) was used to calculate and determine the reduction of viral RNA level in both cell supernatant and cell lysate isolated from both cell lines.
Results: The sequence alignment resulted in the selection of highly conserved regions. The orf1ab region was used to predict and design the siRNAs and a total of twenty-one siRNAs were finally selected from four hundred and twenty-six siRNAs generated by online software. Inhibition of viral replication and significant reduction of viral RNA was observed against selected siRNAs in both cell lines at both concentrations. Based on the Ct value, the siRNAs # 11, 12, 18, and 20 were observed to be the best performing in both cell lines at both concentrations.
Conclusion: Based on the results and data analysis, it is concluded that the use of two different transfection reagents was significantly effective. But the Lipofectamine™ 2000 was found to be a better transfection reagent than the JetPRIME[SUP]R[/SUP] for the delivery of siRNAs in both cell lines.
Keywords: HEK-293-T cells; In silico prediction; MERS-CoV; MERS-CoV, Middle East respiratory Syndrome Coronavirus; Saudi Arabia; Vero Cells; qRT-PCR, quantitative Real-Time PCR; siRNAs; siRNAs, short interfering RNAs.