Rapidly Optimize Anti-miR™ Inhibitor Transfection Using Real-Time RT-PCR
The let-7c/HMGA2 model system is highly useful as a positive control for any Anti-miR experiment and can also be used to optimize conditions used to deliver Anti-miR Inhibitors into cells of interest (Figure 1A). Because let-7c and HMGA2 are ubiquitously expressed, this system can serve as a universal positive control in a variety of human, mouse, and rat cell types, including Hep-G2, A549, ME180, UMR106, and Hepa 1-6. The upregulation of HMGA2 appears to be specific since it is not observed in similar transfections using the Anti-miR-16 Inhibitor (Figure 1B).
Figure 1. Detection of let-7c Anti-miR™ Inhibitor-mediated Upregulation of HMGA2 mRNA Using a TaqMan® Gene Expression Assay (Assay ID: Hs00971725). (A) Optimization experiment: HeLa cells (6 x 103 cells/well; quadruplicate samples) were transfected with let-7c Anti-miR Inhibitor (50 nM) using 0–0.7 µL siPORT™ NeoFX™ Transfection Agent (Cat# AM4510). HMGA2 mRNA levels were measured using real-time RT-PCR 24 hours later. The observed CT values were normalized with results from the 18S rRNA endogenous control. The maximal upregulation of HMGA2 by let-7c Anti-miR Inhibitor (relative to cells transfected Anti-miR Inhibitor—Negative Control #1) is observed using 0.3 µL siPORT NeoFX Agent per well. No cytotoxicity is observed under these conditions. (B) HeLa cells were transfected in triplicate with 50 nM Anti-miR Inhibitors as described for Panel A. Upregulation of HMGA2 mRNA is specifically caused by transfection of let-7c Anti-miR Inhibitor but not by Anti-miR-16 or Anti-miR Inhibitor—Negative Control #1.
Sarah LaMartina, Tera Schaller, and Joe Krebs • Applied Biosystems, Austin, TX
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