7900 quantitative pcr system

Total RNA was isolated from ESCC cells or xenograft tumors, and 1 microgram of total RNA was used for reverse transcription. Quantitative real-time PCR was performed in triplicate on an Applied Biosystems 7900 quantitative PCR system (Foster City, CA, USA). Results were analyzed using the 2^−ΔΔct method with GAPDH as the internal reference genes.

Fecal genomic DNA was extracted using a QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s instructions. Real-time PCR was performed to detect the P. copri level using 20 ng of genomic DNA on an Applied Biosystems 7900 quantitative PCR system. P. copri quantitation was measured relative to the 16s rDNA gene.

Total RNA was extracted by Trizol (Invitrogen, USA), and cDNA was synthesized using the PrimeScript TM RT Reagent Kit (Perfect Real Time, TaKaRa, Japan). Quantitative real-time PCR was performed in a 10ul total volume containing SYBR Green (SYBR® Premix Ex Taq TM II, TaKaRa, Japan) on an Applied Biosystems 7900 quantitative PCR system. The primers used were as follows: LRG1, 5′-GTTGGAGACCTTGCCACCT-3′ and 5′-GCTTGTTGCCGTTCAGGA-3′; HIF-1α, 5′-TGCTAATGCCACCACTACC-3′ and 5′-TG ACTCCTTTTCCTGCTCTG-3′; VEGF-A, 5′-CTTTCTGCTGTCTTGGGTG-3′ and 5′-ACT TCGTGATGATTCTGCC-3′; Twist1, 5′-AGTCCGCAGTCTTACGAGGA-3′ and 5′-GCCAG CTTGAGGGTCTGAAT-3′; E-cadherin, 5′-TACACTGCCCAGGAGCCAGA-3′ and 5′-TGG CACCAGTGTCCGGATTA-3′; N-cadherin, 5′-TTTGATGGAGGTCTCCTAACACC-3′ and 5′-ACGTTTAACACGTTGGAAATGTG-3′; VDR, 5′-GATGCCCACCACAAGACCTA-3′ and 5′-CGGTTCCATCATGTCCAGTG-3′; Vimentin, 5′-TGAGTACCGGAGACAGGTGCA G-3′ and 5′-TAGCAGCTTCAACGGCAAAGTTC-3′; α-SMA, 5′-CGTGGCTACTCCTTCGT G-3′ and 5′-TGATGACCTGCCCGTCT-3′; β-actin, 5′-TGGCACCCAGCACAATGAA-3′ and 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. Relative expression of each specific gene was determined in accordance with the 2^−ΔΔCt method, using β-actin as the internal standard. Each experiment was performed as triplicate, and data was presented as mean ± SEM.