Abi 7900ht real time pcr instrument

RNA was extracted from liver tumors or normal livers with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). RNA extraction, cDNA synthesis and qPCR were performed according to the manufacturer's protocols. Initially, the RNA samples were screened based on their purity (260/280 ≥1.8) with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized via Superscript III, using 5 µg of RNA and oligo-dT as primer. RT-qPCR was performed on an ABI 7900HT real-time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) with primers as follows: GAPDH (NM_017008.4, 5′-AGTGCCAGCCTCGTCTCATA-3′, 5′-TACGGCCAAATCCGTTCACA-3′), GRP78 (NM_013083.2, 5′-TCGACTTGGGGACCACCTAT-3′, 5′-GCGGCCGTTCTTGAATACAC-3′), GRP94 (NM_001012197.2, 5′-TAAGCTCTATGTGCGCCGAG-3′, 5′-TCACGGGAAACATTGAGGGG-3′), ATF4 (NM_024403.2, 5′-TTAAGCCATGGCGCTCTTCA-3′, 5′-GACATTAAGTCCCCCGCCAA-3′), PDIA4 (NM_053849.1, 5′-AGTGGAGAGGACGTCAATGC-3′, 5′-CCCTGACTGGTCCCTTGTTG-3′), ERDJ4 (NM_012699.2, 5′-AACAGGACGAAGGTTGCTCG-3′, 5′-AACTGACTGTGGAGTTGCCA-3′) and GADD34 (NM_133546.3, 5′-GAGAATGTGGCCCCAGTTGA-3′, 5′-ACAATGCTGGGTACTCTGGC-3′). The cycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of primer annealing at 60°C for 30 sec and an extension of amplicon at 72°C for 1 min. The RNA levels were quantified using the 2^−ΔΔCq method (26 (link)).