3/27/2023 0 Comments Flowjo download![]() ![]() MiRNAs have preferential conserved interaction with most human mRNAs, and thereby significantly contribute to almost every developmental and physiological process. MiRNAs are small, non-coding RNAs that are critical for post-transcriptional regulation of gene expression. Furthermore, our findings suggest that LNA-PS miRNA inhibitors can be delivered in the absence of a lipid-based carrier, whereas miRNA mimics need the aid of a lipid-based carrier to achieve sufficient cellular uptake. LNA miRNA inhibitors efficiently downregulated the cellular expression of miRNA, such as miR-15a-5p. None of the miRNA mimics effectively induced overexpression of the respective miRNA when given to primary cells without a carrier. When using a carrier, mirVana and LNA miR-15a-5p and miR-20b-5p mimics showed similar efficiency 48 h following transfection to ECs and monocytes. ![]() Interestingly, LNA-PS miR-15a-5p inhibitor efficiently reduced the levels of miR-15a-5p when delivered without a lipid-based carrier in both ECs and monocytes. MirVana miR-15a-5p inhibitor displayed a less efficient inhibitory effect, which was not improved 48 h following a single transfection or two consecutive transfections. Transfection of LNA inhibitors with either phosphodiester (PE)- or phosphorothioate (PS)-modified nucleotide bonds, delivered using a lipid-based carrier, efficiently downregulated the expression levels of miR-15a-5p already 24 h following transfection. We systematically examined and optimized the transfection conditions of such miRNA inhibitors and mimics to primary endothelial cells and monocytes using either a lipid-based carrier (lipofectamine) for delivery or an unassisted uptake. MiRNA inhibitors and mimics from two commonly used commercial vendors were employed, i.e., mirVana (Thermo Fisher Scientific) and locked nucleic acid (LNA) miRNA (Qiagen). Here, we aimed to investigate how various conditions affect the transfection efficacy of two miRNAs with high and low endogenous expression, miR-15a-5p and miR-20b-5p respectively, in human primary cells. Inhibitors and mimics of miRNAs with a unique chemistry and/or structural modifications are available commercially and require different transfection conditions. A comprehensive dissection of the role of microRNAs (miRNAs) in gene regulation and subsequent cell functions requires a specific and efficient knockdown or overexpression of the miRNA of interest these are achieved by transfecting the cell of interest with a miRNA inhibitor or a miRNA mimic, respectively. ![]()
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