A3500 rt pcr system

Total RNA extraction of 162 pairs of frozen specimens or cultured colon cancer cells was performed according to the manufacturer's instructions (AllPrep DNA/RNA Mini Kit, Qiagen, Germany). The RNA concentration and purity were measured by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, USA). First-strand cDNA was synthesized from one microgram of total RNA using the A3500 RT-PCR System (Promega, USA). Quantitative real-time PCR was performed on a Mastercycler® ep realplex (Eppendorf, Germany) with a SYBR Green RNA PCR kit (Fermentas, USA) according to the manufacturer's protocol. GRK3 was amplified with the following primers: 5′-gcagtgccgactggttct-3′ (forward primer) and 5′-gtctgaaagggctgtgacct-3′ (reverse primer); β-actin was used as an internal control with the following primers: 5′-cgggaaatgtgcgtgac-3′ (forward primer) and 5′-tggaaggtggacagcgagg-3′ (reverse primer). Each reaction was run in triplicate. The relative GRK3 mRNA expression was calculated using 2^−△△Ct comparative method.

Total RNA of frozen tissues was extracted using an All-Prep DNA/RNA Mini kit (Qiagen, Hilden, Germany). cDNA was reverse transcribed with the A3500 RT-PCR System (Promega Corporation, Madison, WI, USA) from 1 μg of total RNA. The relative TXNDC9 mRNA levels were detected by qPCR with Mastercycler^® ep realplex (Eppendorf, Hamburg, Germany) using an iQ^TM SYBR^® Green Supermix kit (Bio-Rad, Hercules, CA, USA). The mRNA levels were normalized to those of β-Actin. The following specific primers for qPCR were used: TXNDC9-F: 5′- CTGCTTCAGACTACCAAACTGG-3′, TXNDC9-R: 5′- CTCTGTAGAAATGGCAAACCACA-3′; SRM-F: 5′-GTGGTGGCCTATGCCTACTG-3′, SRM-R: 5′-CTCCTGGAAGTTCGTGCTCG-3′; DUSP2-F: 5′-GGGCTCCTGTCTACGACCA-3′, DUSP2-R: 5′-GCAGGTCTGACGAGTGACTG-3′; WDR74-F: 5′-CCTGGGGTGTGTAGGATGC-3′, WDR74-R: 5′-CAAGTCCAGCCAGTCATTCCG-3′; PES1-F: 5′-GGCCACCAACTACATCACCC-3′, PES1-R: 5′-AGAATGCACAGCCGCCTAAA-3′; β-Actin-F: 5′- CATGTACGTTGCTATCCAGGC-3′, β-Actin-R: 5′-CTCCTTAATGTCACGCACGAT-3′. The relative mRNA expression levels were evaluated using the 2^−ΔΔCT comparative method.