( r System for quantificationof the gr...)Quantification of siRNA Silencing Efficiency,,,Using the LightCycler System

r System for quantification of the green fluorescent protein (GFP) mRNA level relative to a housekeeping gene mRNA after GFP gene knockdown of a cell with a stably transfected GFP gene. This method was successfully used to show the specificity and silencing efficiency of GFP-specific siRNAs.

Materials and Methods

Cell culture Cells of the human adherent cell line PC3 stably transfected with the gene for the GFP (PC3-GFP) were cultured in RPMI 1640 containing 10% (v/v) fetal calf serum, 2 mM L-glutamine and 750 g/ml neomycin. Two days prior to transfection with siRNA oligonucleotides, 5x104 cells were plated in each well of 24-well plates. The siRNA (Dharmacon) was mixed with diluted transfection reagent and applied to the cells in a 100 nM concentration in medium without serum (Opti-MEM). After 4 hours of transfection, fetal calf serum was added to 10% (v/v) final concentration.

GFP assay GFP concentration was measured in 100 l lysate with a spectrofluorophotometer RF-5301 (wavelength: EX: 395 nm, EM: 508 nm).

Isolation of mRNA The mRNA was isolated with the MagNA Pure LC Instrument using the MagNa Pure LC mRNA Isolation Kit I according to the kit protocol for cultured cells and the MagNA Pure protocol mRNA I cells. Elution volume was 50 l.

cDNA synthesis Using the 1st Strand cDNA Synthesis Kit for RT-PCR with random primers according to the kit protocol, 5 l mRNA of each sample was transcribed into cDNA. The cDNA was diluted tenfold in PCR-grade water and quantified with the LightCycler Instrument.

Quantitative real-time PCR For the GFP and housekeeping gene amplifica
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