Revertra ace qpcr rt kit with sybr green

Total RNA was isolated from colonoscopy biopsy specimens using TRIzol (Invitrogen, CA, USA) according to the manufacturer`s instructions. Reverse transcription was conducted using the Reverse Transcription Kit (Takara, Osaka, Japan). Quantitative real-time PCR (qRT-PCR) was then performed using the ReverTra Ace qPCR RT Kit with SYBR Green (Toyobo, Osaka, Japan) in triplicates on an Applied Biosystems StepOne Real-Time PCR System (Thermo Fisher, MA, USA). With GAPDH as the internal control target, the expression levels were calculated on the basis of the 2^−ΔΔCt method. The primer sequences for CHGB amplification are (forward primer) 5′-CGA​GGG​GAA​GAT​AGC​AGT​GAA-3′ and (reverse primer) 5′-CAG​CAT​GTG​TTT​CCG​ATC​TGG-3′.

qRT-PCR assays were performed with the real-time PCR detection system (Eppendorf) using total RNA and the ReverTra Ace qPCR RT Kit with SYBR green (TOYOBO, Japan). Sequences of the upstream and downstream PCR primers to detect human Hsp70 mRNA used in qRT-PCR were 5′-GCC ACT CTG CTT ATC AAG TTT C-3′ and 5′-CTC CCA ATG TCG TGT CAA AT-3′, respectively. Upstream and downstream primers for human Grp78 mRNA were 5′-AAA GAA ACC GCT GAG GCT TAT-3′ and 5′-CTG AAA CAG TAT GCC GAC AAG-3′, respectively. Upstream and downstream primers for human 18S rRNA were 5′-CAG CCA CCC GAG ATT GAG CA-3′ and 5′-TAG TAG CGA CGG GCG GTG TG-3′, respectively.