Volume 23, Issue 2 (2020)
mjms 2020, 23(2): 67-74 |
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Shafaati M, Jamalidoust M, Kargar M, Arefian E, Kafilzadeh F. Luciferase Assay for Demonstrate the Competence of Selective MicroRNA of let-7b in Suppressing HCV Replication. mjms 2020; 23 (2) :67-74
URL: http://mjms.modares.ac.ir/article-30-41254-en.html
URL: http://mjms.modares.ac.ir/article-30-41254-en.html
1- Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
2- Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran , mjamalidoust@gmail.com
3- Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran
2- Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran , mjamalidoust@gmail.com
3- Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran
Abstract: (2029 Views)
Aims: The development of new antiviral agents is an appropriate approach to eradicate hepatitis C infection. Due to the lack of suitable animal models, there is always a barrier to the proper evaluation of antiviral compounds in vivo. The growing attention to microRNAs is a new strength in antiviral therapy. The aim of the present study was to use luciferase assay to confirm the specific interaction between miRNA and genomic RNA of hepatitis C virus (HCV) genotype 1b to suppress the replication of the virus.
Materials & Methods: The NS5B genomic fragments of the HCV genome were sub-cloned into the psiCHECK-2-TM vector as MRE. The relative expression of lentivirus vectors expressing miRNAs in Huh7.5 cells was assessed through fluorescence microscopy and real-time PCR. The lentivirus expressing let-7b was transduced to Huh7.5 cells. The NS5B-psiCHECK-2-TM (MRE) was transfected to the Huh7.5 cell. The relative expression of luciferase was measured using a luciferase dual assay kit.
Findings: With the use of lentiviruses expressing let-7b, high and permanent expression of let-7b was created in the target cell. On the other hand, the specific attachment of the responsive sequence (NS5B) to the microRNA of let-7b was shown by decreasing luciferase light.
Materials & Methods: The NS5B genomic fragments of the HCV genome were sub-cloned into the psiCHECK-2-TM vector as MRE. The relative expression of lentivirus vectors expressing miRNAs in Huh7.5 cells was assessed through fluorescence microscopy and real-time PCR. The lentivirus expressing let-7b was transduced to Huh7.5 cells. The NS5B-psiCHECK-2-TM (MRE) was transfected to the Huh7.5 cell. The relative expression of luciferase was measured using a luciferase dual assay kit.
Findings: With the use of lentiviruses expressing let-7b, high and permanent expression of let-7b was created in the target cell. On the other hand, the specific attachment of the responsive sequence (NS5B) to the microRNA of let-7b was shown by decreasing luciferase light.
Conclusion: Lentiviral vectors are used to maintain high and stable expression of microRNAs in cells. The use of luciferase assay is one of the most appropriate methods to confirm the interaction between miRNA-mRNA that can be used for other viral genes with different microRNAs.
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