Transcriptor first strand cdna synthesis kit with random hexamer

Quantitative RT-PCR (qRT-PCR) was performed as previously described [45 (link), 46 ]. Total RNA was extracted from exponentially growing cells using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit with random hexamer (Roche). qRT-PCR was carried out using Power SYBR Green PCR Master Mix (Life Technologies) on 7900HT Fast Real-Time PCR System (Life Technologies) according to the manufacturer’s instructions. cDNA equivalent to 10 ng RNA was amplified with 75 nM forward and 75 nM reverse primers in 10 µL reaction. Dissociation curves were performed to confirm specific amplifications without primer dimer formation. Samples were also subjected to gel electrophoresis analysis to confirm that the PCR products were of expected size. Abundance of mRNA expression was determined using the comparative C_T method and expressed relative to ACTB expression.

Quantitative RT-PCR was performed as previously described [59 (link)]. Total RNA was extracted from exponentially growing cells using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit with random hexamer (Roche). qRT-PCR was carried out using Power SYBR Green PCR Master Mix (Life Technologies) on 7500 Fast Real-Time PCR System (Life Technologies) according to the manufacturer’s instructions. cDNA equivalent to 10 ng RNA was amplified with 75 nM forward and 75 nM reverse primers in 10 μL reaction. Dissociation curves were performed to confirm specific amplifications without primer dimer formation. Samples were also subjected to gel electrophoresis analysis to confirm that the PCR products were of expected size. Abundance of mRNA expression was determined using the comparative C_T method and expressed relative to ACTB expression. The following cycling conditions were used: 1 cycle of 95 °C for 10 min, 50 cycles of 95 °C for 15 s and 60 °C for 1 min. The following primers were used for the detection of mRNA expression: ACTB-F: 5′- CACCTTCACCGTTCCAGTTT-‘3; ACTB-R: 5’-GATGAGATTGGCATGGCTTT-3′; Chk/Matk-F: 5′-TCGTGTTGCATCTTCGTCAT-‘3; Chk/Matk-R: 5’-CACAGATCGGAGAGGGAGAG-‘3; Csk-F: 5’-CACAGATCGGAGAGGGAGAG-‘3; Csk-R: 5’-CTGACCGCATGGACCGT-‘3.