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Tech Note

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, Loma Linda University (Center for Health Disparities & Molecular Medicine)

Introduction Results Conclusions Methods

Introduction  

RNA interference (RNAi) 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 transfection 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 PSIP1, the gene encoding LEDGF/p75 (lens epithelium derived growth factor, 75 kDa) in docetaxel-resistant prostate cancer PC-3-DR cells.

Results  

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 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 the PISP1 gene (199 bp). The data confirmed a 54.5% knockdown of LEDGF/p75 expression in PC-3-DR cells transfected with siRNA against PISP1 compared to cells transfected with control scrambled siRNA.

Real-time PCR amplification curves for GAPDH and RNase P

Figure 1. Measurement of LEDGF/p75 knockdown by qPCR. The bar graphs indicate normalized expression levels for LEDGF/p75 mRNA in the presence of negative control (control) or targeted siRNA constructs (knockdown).

 

qPCR Data
Symbol Control Knockdown ΔCt K.D ΔCt con ΔΔCt 2^(– ΔΔCt) Up/Down
Well 1 Well 2 Well 3 AVG Well 1 Well 2 Well 3 AVG GOI-HKG GOI-HKG KD-Con Fold Regulation
LEDGF/p75 23.4 24.38 24.03 23.937 18.88 21.77 19.79 20.15 7.44 6.31 1.137 0.455 –2.199
GAPDH 15.68 16.23 17.57 16.493 14.02 13.73 13.77 13.84 0.00 0.00 0.000

Figure 2. qPCR Data.

Conclusions  

Under the conditions tested, good yields 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 are particularly notable.

Methods  

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-min RT step.

After the RT reactions were complete, 1 µl of each RT reaction was used as template in a 25-µl 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 for 15 sec and 60°C for 1 min. A MyiQ system (Bio-Rad) was used for real-time PCR.

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Cat. # Product Size Price License Quantity Details
RR036A PrimeScript™ RT Master Mix (Perfect Real Time) 200 Rxns USD $507.00

This product is a reverse transcription reagent kit designed to perform reverse transcription optimized for two step real-time RT-PCR (RT-qPCR). It contains a 5X pre-mixed reagent containing all of the components needed for quantitative RT-PCR reverse transcription (PrimeScript RTase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP Mixture, and reaction buffer), and a reaction can be started simply by adding template RNA and water. Because it uses PrimeScript RTase, which features excellent extension, this product makes it possible to synthesize template cDNA for real-time PCR efficiently in a short time. The cDNA obtained with this product can be used in both intercalating green dye qPCR assays and probe qPCR assays.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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RR036A: PrimeScript RT Master Mix (Perfect Real Time)

RR036A: PrimeScript RT Master Mix (Perfect Real Time)
RR036B PrimeScript™ RT Master Mix (Perfect Real Time) 800 Rxns USD $1625.00

This product is a reverse transcription reagent kit designed to perform reverse transcription optimized for two step real-time RT-PCR (RT-qPCR). It contains a 5X pre-mixed reagent containing all of the components needed for quantitative RT-PCR reverse transcription (PrimeScript RTase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP Mixture, and reaction buffer), and a reaction can be started simply by adding template RNA and water. Because it uses PrimeScript RTase, which features excellent extension, this product makes it possible to synthesize template cDNA for real-time PCR efficiently in a short time. The cDNA obtained with this product can be used in both intercalating green dye qPCR assays and probe qPCR assays. Cat. # RR036B contains 4 of Cat. # RR036A. Please refer to Cat. # RR036A for complete product documentation and resources.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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