Transfecting Colon Cancer HCT-116 Cells with RJH Reagents to silence Polynucleotide Kinase 3'-Phosphatase (PNKP) Expression
Colon cancer HCT-116 cells (ATCC) were seeded and grown in 6-well plates overnight before transfection with siRNA complexes. A specific siRNA against Polynucleotide Kinase 3'-Phosphatase (PNKP; obtained from IDT) was formulated with LipofectamineTM 2000 and the RJH transfection reagents, as recommended by the respective manufacturers (see below for specific siRNA:transfection reagent ratios used). As a control, a scrambled siRNA was also formulated with the same reagents and used in cell treatments. The cells were transfected in complete tissue culture medium (DMEM + 10% FBS, 1% Pen/Strep) at siRNA concentration of 50 nM, and allowed to incubate with the formulated siRNAs for additional 2 days. The cells were harvested and processed for western blot analysis according to the established procedures. The blots were stained with an in-house generated polyclonal antibody and visualized using chemoluminescence.
The indicated lanes in the western blot correspond to the following treatments:
1) untreated cells
2) cells exposed to scrambled siRNA/Lipo-2000 (1:2)
3) cells exposed to PNKP siRNA/Lipo-2000 (1:2)
4) cells exposed to scrambled siRNA/Lipo-2000 (1:4)
5) cells exposed to PNKP siRNA/Lipo-2000 (1:4)
6) cells exposed to PNKP siRNA/ALL-Fect (1:6)
7) cells exposed to PNKP siRNA/293-Fect (1:6)
8) cells exposed to PNKP siRNA/Prime-Fect (1:6)
9) cells exposed to scrambled siRNA/ALL-Fect (1:6)
10) cells exposed to siRNA/293-Fect (1:6)
11) cells exposed to scrambled siRNA/Prime-Fect (1:6)
The lanes treated with the scrambled siRNA (#2, 4, 9, 10, and 11) gave band intensities equivalent to the untreated cells (#1), irrespective of the transfection reagent used. With PNKP specific siRNA treatments, significant reductions in the band intensities were evident; LipofectamineTM 2000 at ratio of 1:4 was more potent than 1:2 (siRNA:reagent respectively). All 3 RJH reagents were effective in reducing the PKNP protein levels, indicating the versatility of the reagents. Note that highest silencing was seen with 293-Fect in this study. All 3 RJH reagents, ALL-Fect, 293-Fect and Prime-Fect were effective in silencing the chosen target by 80-90%, based on semi-quantitative densitometric analysis.
| Data courtesy of I. Paiva, University of Alberta (Canada)|