Transfection of microRNA (miRNA) mimics is increasingly being used to examine biological effects of specific miRNAs on cell function. For these gain-of-function experiments, use the Pre-miR™ miRNA Starter Kit to optimize miRNA mimic transfection into cultured cells (see sidebar). Real-time PCR detection of the downstream target of the hsa-miR-1 miRNA mimic (positive control included in the kit) is the preferred method for assessing transfection efficiency of this control. However, most other miRNAs down regulate target gene expression at the protein level. Here, we show how Western blots performed using the Western-Star™ Immunodetection System can be used to detect Pre-miR hsa-miR-1 miRNA Precursor induced knockdown of PTK9 at the protein level.

Gain-of-Function Experiments with Pre-miR miRNA Precursors

Pre-miR miRNA Precursors are small, chemically-modified, double-stranded RNA molecules, which mimic endogenous mature miRNA molecules. These ready-to-use miRNA mimics are introduced into cells using simple transfection or electroporation techniques. They are then used to measure the biological function of specific miRNAs in cells, thus allowing detailed study of miRNA effects via gain-of-function experiments.

Detecting Effects of an miRNA Positive Control

Until recently, an miRNA positive control, whose affects are easily monitored, has not been available. The Pre-miR hsa-miR-1 miRNA Precursor can now be used for this purpose and is the positive control included in the Pre-miR miRNA Starter Kit. This human miR-1 miRNA mimic effectively down-regulates the expression of twinfilin-1 (also known as PTK9) by 60–95% at the mRNA level. Effective delivery and activity of Pre-miR hsa-miR-1 miRNA Precursor (positive control) can be detected by real-time PCR using TaqMan® Gene Expression Assays for PTK9 (Figure 1). The shift in the PTK9 plots between the positive control transfected samples and the Pre-miR miRNA Precursor—Negative Control #1 (negative control) transfected samples represents an 89% knockdown of PTK9 mRNA.

Figure 1. Knockdown of PTK9 mRNA by Transfection of Pre-miR™ hsa-miR-1 miRNA Precursor. HeLa cells were transfected in 96-well plates (4000 cells/well) with 50 nM Pre-miR hsa-miR-1 miRNA Precursor (positive control) or Pre-miR miRNA Precursor—Negative Control #1 (negative control) using siPORT™ NeoFX™ Transfection Agent. Two days post-transfection, RNA was isolated (MagMAX™-96 Total RNA Isolation Kit) and reverse transcribed (RETROscript® Kit). Relative quantification of PTK9 mRNA was performed using TaqMan® Gene Expression Assays (PTK9 Assay ID: Hs00702289_s1). The TaqMan Assay targeting 18S rRNA (Assay ID: Hs99999901_s1) is used to normalize differences in RNA input. Shown above are the amplification plots for PTK9 and 18S rRNA in positive and negative control transfected samples.

Although the functional effects of the miR-1 miRNA mimic are readily detected at the mRNA level using the supplied PTK9 TaqMan Assay, the effects of the positive control could also be observed at the protein level by immunoblotting (Figure 2). Positive and negative control Pre-miR miRNA Precursors were transfected in triplicate into HeLa cells using siPORT™ NeoFX™ Transfection Agent in 6-well plates. Samples were harvested after three days, lysed, and separated by electrophoresis. The proteins were transferred to a PVDF membrane, and the membrane was first incubated with an a-PTK9 antibody and then reassessed for GAPDH as a loading control. The Applied Biosystems Western-Star™ Immunodetection System was used to detect the PTK9 protein (see sidebar, Highly Sensitive Chemiluminescent Immunodetection). As shown in Figure 2, PTK9 protein levels were greatly reduced in positive control transfected samples relative to negative control transfected samples. This is consistent with the robust knockdown of PTK9 mRNA detected by TaqMan Assay shown in Figure 1. These results demonstrate that the Western-Star Immunodetection System is ideal for use with the Pre-miR miRNA Starter Kit to detect knockdown of PTK9 (the target of the Pre-miR hsa-miR-1 miRNA Precursor included in the kit) at the protein level.

Figure 2. Decrease in PTK9 Protein Levels by Transfection of Pre-miR™ hsa-miR-1 miRNA Precursor. HeLa cells were reverse transfected in 6-well plates (180,000 cells/well) with 50 nM Pre-miR™ hsa-miR-1 miRNA Precursor or Pre-miR miRNA Precursor—Negative Control #1 (Pre-miR Negative Control) using siPORT™ NeoFX™ Transfection Agent (1.5 µL/well). Cells were harvested after 3 days and lysed in 100 µL Laemmli Protein Sample Buffer. Then the sample (20 µL) was separated by SDS-PAGE and transferred to PVDF membrane. The blot was incubated with a-PTK9 antibody (1:1000 dilution; Abnova Corp, Cat#H00005756-M05) and developed with the Western-Star™ System. Shown above are transfection triplicates. The blot was reassessed for GAPDH as a loading control.