Monitoring siRNA knockdown: Quantifying transient knockdown of LEDGF/p75 in docetaxel-resistant prostate cancer PC-3-DR cells
Data kindly provided by: Anamika Basu, Scientist at Loma Linda University (Center for Health Disparities & Molecular Medicine)
RNAi (RNA interference) may be used to degrade a specific target mRNA, thereby diminishing the production of protein encoded by the targeted transcript in order to understand its function. Transient tranfection of cell lines with short interfering RNA (siRNA) molecules can be a rapid technique to allow analysis of the biological consequences of reducing a protein target to low or trace amounts. Quantitative monitoring of such experiments at the RNA level requires the synthesis and quantification of cDNA corresponding to the targeted transcript with great sensitivity.
The objective of this experiment was to quantify the production of cDNA products using PrimeScript RT Master Mix (Perfect Real Time) to assess the transient knockdown of the gene encoding LEDGF/p75 (lens epithelium derived growth factor of 75 kDa) in docetaxel-resistant prostate cancer PC-3-DR cells.
PC-3-DR cells were transfected with either scrambled sequence siRNA (control) or siRNA designed to result in transient knockdown of LEDGF/p75 (PSIP1). Extracted RNA was reverse transcribed using PrimeScript RT Master Mix according to the standard protocol. cDNA was used for qPCR to quantify the transient LEDGF/p75 depletion.
The results (Figure 1) indicated that cDNA products were obtained from the reverse transcription reaction, and that these products could be analyzed by qPCR with primers specific for LEDGF/p75 gene (amplicon size 199 bp). The data confirmed a 54.5% knockdown of LEDGF/p75 in PC-3-DR cells transfected with siRNA against LEDGF/p75 compared to cells transfected with control scrambled siRNA.
Figure 1. Real-time PCR amplification curves for GAPDH and RNase P. The blue curves are derived from cDNA templates generated with the PrimeScript RT Kit, and the red curves are from the Company I kit.
Table 1. qPCR Data
Under the conditions tested, good yield of cDNA products were obtained with PrimeScript RT Master Mix (Perfect Real Time), allowing effective monitoring of siRNA knockdown. The consistent Ct values obtained using this master mix were particularly notable.
The docetaxel-resistant cell line used for this experiment was PC-3-DR. Cells were transiently transfected with either scrambled sequence siRNA or a siRNA designed to target the PSIP1 gene encoding LEDGF/p75. Following transfection,RNA was extracted from control or experimental cells. Total RNA (0.25 µg) was used for reverse transcription reactions with PrimeScript RT Master Mix (Perfect Real Time), using the standard protocol described in the product User Manual with a 15-minute RT step.
After the RT reactions were complete, 1 µl of each RT reaction was used as template in a 25 µl-volume qPCR with iQ SYBR® Green Supermix (Bio-Rad). PCR cycling conditions were as follows: 95°C 10 min followed by 40 cycles of 95°C, 15 sec, 60°C, 1 min. A MyiQ system (Bio-Rad) was used for real-time PCR.