Luciferase reporter assay for miRNA targets

Genomic DNA were isolated from freshly prepared PBMCs using QIAamp DNA mini kit (Qiagen) according to manufacturer’s instructions. The 3′UTRs of predicted miRNA target genes were PCR amplified using Phusion Taq polymerase (NEB, Ipswich, MA, USA). The amplified products were digested with restriction enzymes (Xho I and Not I) and ligated downstream to the luciferase reporter gene in psiCHECK™-2 vector (Promega). Dual luciferase assays were performed as described earlier (26 (link), 28 (link), 30 (link)).

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Naqvi A.R., Shango J., Seal A., Shukla D, & Nares S. (2018). Viral miRNAs Alter Host Cell miRNA Profiles and Modulate Innate Immune Responses. Frontiers in Immunology, 9, 433.

Publication 2018

QIAamp DNA mini kit Phusion Taq polymerase psiCHECK™-2 vector

Assays Genes Genomic Luciferase Mirna Promega Reporter gene Restriction enzymes Taq polymerase Utrs Vector

Corresponding Organization : University of Illinois Chicago

Top 5 similar protocols

Variable analysis

independent variables

DNA isolation using QIAamp DNA mini kit
PCR amplification of 3'UTRs of predicted miRNA target genes using Phusion Taq polymerase
Digestion of amplified products with restriction enzymes (Xho I and Not I)
Ligation of digested products downstream to the luciferase reporter gene in psiCHECK™-2 vector

dependent variables

Luciferase reporter gene expression (measured by dual luciferase assays)

control variables

Not explicitly mentioned

controls

Positive controls: Not specified
Negative controls: Not specified

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