Construction of the eukaryotic expres ion vector for positive control plasmid of hum an MicroRNA-335 and it′s significance
WEN Zhi-hong
DAI Yan
HE Shuan g
Abstract：Objective To construct the eukaryotic expression vector for the positive control plasmid of has-miR-335.Me thods One pair of complementary oligomeric chains of exact matching sequence for has-miR-335 seed region were synthesized,and then target fragments were obtained by annealing.The target fragments were inserted into pMIR-REPORT luciferase reporter vector.We cotransfected 293T/17 cell with the recombinant plasmid or pMIR-RE-PORT plasmid together with Pre-miRTMmicroRNA 335 Precursor by Lipofectamine 2000.The expression of has-miR-335 in eukaryotic cells was tested by Luciferase Assay.Results The positive control recombinant plasmid of hsa-miR-335 was comfirmed by sequencing.The expression of has-miR-335 decreases after combined with it′s positive control in eukaryotic cells.Conclusion The positive control plasmid for has-miR-335 can be used to confirm if the methods of experiments in RNA extraction,gene transfection and expression are reliable.The double luciferase reporter gene detection system of has-miR3-35 is constructed successfully,which lays the foundation for testing and validation of has-miR-335 target genes.
Keywords：has-miR-335Positive controlPlasmidLuciferase reporter vectorpMIR-REPORT
Publication Date：2015-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：3( 1-3 )