Getting Started with miRNA Analysis Experiments
Figure 1. Quantitation of PTK9 mRNA Down-regulation in HeLa Cells. After transfection of Pre-miR™ hsa-miR-1 Precursor HeLa cells (6,000 cells/well) were transfected in 96-well plates with either 50 nM miRNA Precursor miR-1 Pre-miR miRNA Precursor or 50 nM Pre-miR Negative Control #1 using siPORT™ NeoFX™ Transfection Agent. Cells were harvested 2 days post-transfection. PTK9 Ct values were normalized using Ct values for 18S rRNA from the corresponding samples. (A) qRT-PCR reactions showed a DCt shift caused by Pre-miR hsa-miR-1 Precursor transfection, which corresponds to a >90% reduction in PTK9 mRNA levels compared to cells transfected with Pre-miR Negative Control #1 (B).
The positive control miRNA mimic provided in the kit, Pre-miR hsa-miR-1 Precursor, down regulates PTK9 mRNA , enabling scientists to test and confirm delivery of Pre-miR miRNA Precursors in the cell line of choice. Successful delivery of this miRNA causes a dramatic decrease (60-95%) in intracellular levels of PTK9 mRNA in a wide variety of cell lines (Figure 2).
Figure 2. miR-1 Mediated Knockdown of PTK9 mRNA in Various Human Cell Lines. Human cell lines were transfected in 96-well plates in triplicate with either 50 nM Pre-miR™ hsa-miR-1 miRNA Precursor or Pre-miR Negative Control #1 using 0.3 µL/well siPORT™ NeoFX™ Transfection Agent. For each transfection condition, the amount of PTK9 mRNA in the miR-1 transfected cultures was compared to that in the negative control transfected samples. In every case, transfection of miR-1 Pre-miR precursor caused a >60% decrease in PTK9 expression.
The Pre-miR miRNA Precursor Starter Kit can be used to optimize transfection of Pre-miR miRNA Precursors into the cell line of choice (Figure 3). Once identified, these transfection conditions can be used for all subsequent miRNA transfections in that cell type. A suggested optimization procedure is outlined in the Instruction Manual for the kit.
Figure 3. Effect of siPORT™ NeoFX™ Transfection Agent Concentration on Pre-miR™ hsa-miR-1 miRNA Precursor Mediated Knockdown of PTK9 Expression in HeLa Cells. The Pre-miR miRNA Precursor Starter Kit can be used to optimize transfection of Pre-miR miRNA Precursors in cells. HeLa cells (4000 cells/well) were treated (in triplicate) with either 50 nM Pre-miR hsa-miR-1 miRNA Precursor or Pre-miR Precursor Negative Control #1 in conjunction with varying volumes of siPORT NeoFX Transfection Agent. For each transfection condition, the amount of PTK9 mRNA in the Pre-miR hsa-miR-1 miRNA Precursor transfected cultures was compared to that in the negative control transfected samples. In this experiment, the optimal siPORT NeoFX Transfection Agent amount was 0.4 µL/well.
Joe Krebs, Luis Foncerrada, Sarah LaMartina, Tera Schaller • Applied Biosystems, Austin, TX