Quantitative Analysis of Gene Expression

Cells treated with arecoline for specific periods of time and biopsy samples from patients (n = 5) were used for RT-PCR analysis. Total RNA was purified from samples using TRIzol^® according to the manufacturer’s instructions. For mRNA analysis, complementary DNA of each sample was randomly primed from 1 μg of total RNA using Superscript-RT. Real time PCR was performed using the KAPA SYBRFAST qPCR KIT. Data was normalized to18s rRNA and relative expression levels were determined using StepOne Real Time PCR software. Semi-quantitative PCR analysis was carried out in a total volume of 10 μl containing 0.5 picomoles of each primer using Go Taq Flexi DNA Polymerase on the 2720 Thermal Cycler (Applied Biosystems, USA) The relative quantification value for each target gene was expressed as 2^−ΔΔCT [49 (link)].