Gotaq rt qpcr master mix system

mRNA was extracted using RNeasy® Plus Mini Kit (Qiagen, Valencia, CA) and cDNA prepared using the GoScript™ Reverse Transcription System (Promega, Madison, WI) with a 1:1 mixture of random and Oligo (dT)₁₅ primers according to manufacturer’s instructions. All RT-qPCR reactions were performed using the GoTaq® RT-qPCR Master Mix System (Promega). Validated primers were purchased from Qiagen Inc. (Valencia, CA): human Cyp1b1 – QT00209496, Cyp1a1 – QT00012341, Twist1 – QT00011956, Snai1 – QT00010010, Snai2 – QT00044128, VIM – QT00095795, Twist2 – QT02454004, FN1 – QT00038024, Notch1 – QT01005109, Notch2 – QT00072212, Aldh1a1 – QT00013286, Aldh1a3 – QT00077588, Pou5f1 – QT00210840, Sox – QT00237601, Nanog – QT01844808, Dppa3 – QT01667197, Msi1 – QT00025389, Human Bmi1 – QT00052654, Tgfb1 – QT00000728, Ahr – QT02422938, and Gapdh – QT01192646. RT-qPCR reactions were performed using a 7900HT Fast Real-Time PCR instrument (Applied Biosystems, Carlsbad, CA), with hot-start activation at 95 °C for 2 min, 40 cycles of denaturation (95 °C for 15 sec), and annealing/extension (55 °C for 60 sec). Relative gene expression was determined using the Pfaffl method [103 (link)] and the threshold value for Gapdh mRNA was used for normalization.