Conveniently Monitor miRNA Transfection and Function at the Protein Level
Gain-of-Function Experiments with Pre-miR miRNA Precursors
Detecting Effects of an miRNA Positive Control
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.
The Pre-miR miRNA Starter Kit contains all the reagents necessary to perform a basic experiment to deliver and measure the biological effects of functional miRNA precursors (all reagents are also available separately):
- Pre-miR hsa-miR-1 miRNA Precursor—a synthetic miRNA mimic that specifically down-regulates the PTK9 gene in human and mouse cells and serves as the positive control.
- Pre-miR miRNA Precursor–Negative Control #1—a nontargeting miRNA mimic.
- TaqMan® Gene Expression Assay* (Assay ID: Hs00702289_s1, Part Number 4331182)—an assay to detect human PTK9 mRNA by real-time RT-PCR. PTK9 mRNA is down-regulated by the positive control.
- siPORT™ NeoFX™ Transfection Agent—a high-quality, lipid-based transfection agent with low cytotoxicity, optimized for delivering siRNAs and miRNA mimics into adherent mammalian cells.
- Nuclease-free Water
*Note: Because the human miR-1 sequence is identical to that of mouse, this control miRNA mimic can also be used in mouse cells. The corresponding TaqMan Gene Expression Assay for mouse PTK9 mRNA is available separately (Assay ID Mm01598980_g1).