HeLa S3 cells (6 x 104 cells/well) were transfected using HiPerFect Transfection Reagent with miScript miR-16 Inhibitor or a negative control. Endogenous miR-16 levels were quantified by real-time PCR using the miScript System. U6B was used as the reference RNA for data normalization and miR-16 levels were expressed as 2-ΔΔCT values relative to the miR-16 levels in untransfected cells. [A] Transfection of miScript miR-16 Inhibitor strongly reduced the amount of detectable endogenous miR-16. [B] The amplification plots indicated a significant decrease in the level of detectable miR-16 as a result of miR-16 Inhibitor transfection. Dissociation curve analysis (inset) reflects the specificity of the real-time PCR detection using the miScript System.
HeLa S3 cells expressmiR-16 at high levels and do not express miR-1 (see Ref. 1). In a cotransfection experiment using HiPerFect Transfection Reagent, HeLa S3 cells (6 x 104 cells/well) were cotransfected with a luciferase reporter construct with an miR-16 binding site in the 3' UTR together with miR-16 Inhibitor. miR-16 Inhibitor was used at varying concentrations in the experiment to evaluate the optimal inhibitor concentration required to see the inhibitory effect. Alternatively, cells were transfected with miR-1 Inhibitor alone as a control. A luciferase construct without an miRNA binding site (WT) was transfected as a positive control. An increase in luciferase expression following transfection of the Inhibitor indicated that the miR-16 Inhibitor prevented endogenous miR-16 from downregulating the reporter gene.
1. Lagos-Quintana, M. et al. (2001) Identification of novel genes coding for small expressed RNAs. Science 294, 853.
HeLa S3 cells (6 x 104 cells/well) were transfected using HiPerFect Transfection Reagent with miR-15a Inhibitor, miR-16 Inhibitor, miR-21 Inhibitor, miR-25 Inhibitor, or an Inhibitor with a scrambled sequence (negative control). At the time points indicated, luciferase reporter constructs with the appropriate miRNA binding sites were transfected. A reporter construct that is not regulated by any miRNA was used as positive control. Twenty-four hours later, luciferase activity was measured.