C1000 thermal cycler cfx 96 real time pcr detection systems

Total RNA were extracted from harvested cells using the Zymo Quick-RNA miniprep extraction kit (Zymo Research, CA, USA) according to the manufacturer's instructions. Quantitative reverse transcription PCR (qRT-PCR) was conducted. For the detection, 1 μg of total RNA per sample was converted to cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). cDNAs were amplified and detected using SYBR Green PCR Kit (Qiagen, Valencia, CA, USA). The GADPH was used as endogenous control for mRNA. For detection of the miRNA, the cDNA products were synthesized using miScript Reverse Transcription Kit (Qiagen, Valencia, CA, USA). The primers specific for miR-let-7a or endogenous control RNU6B were purchased from Qiagen. qRT-PCR was performed using miScript SYBR Green PCR Kit (Qiagen). All reactions were run in triplicate on Bio-Rad C1000 thermal cycler (CFX-96 real-time PCR detection systems, Bio-Rad). The fold change of miRNA or mRNA expression was calculated according to the 2−ΔΔct method. The primers used were as follows: for miR-let-7a forward: 5′-TGAGGTAGTAGGTTGTATAGTTAAA-3′, miR-let-7a reverse: 5′-AACGAGACGACGACAGACTTT-3′; PKM2 forward 5′-GTCGAAGCCCCATAGTGAAG-3′, PKM2 reverse 5′-GTGAATCAATGTCCAGGCGG-3′; and for GAPDH forward, 5′-CATCTTCCAGGAGCGAGA-3′ and GAPDH reverse 5′-TGTTGTCATACTTCTCA-3′ (Invitrogen, Shanghai, China).

Total RNA of the ileal mucosa was extracted using Trizol reagent (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and the integrity was evaluated using agarose–ethidium bromide electrophoresis. Reverse transcription (RT) reactions were immediately performed using the FastQuant RT Kit (Tiangen Biotech Co., Ltd., Beijing, China). Real-time quantitative PCR was conducted in duplicate in the Bio-Rad C1000 thermal cycler (CFX-96 real-time PCR detection systems; Bio-Rad). PCR programs for all genes were as follows: 15 min at 95°C, 40 cycles of 95°C for 10 s, 60°C for 30 s. The relative gene expression levels were calculated using the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)), and avian β-actin was used as reference gene. The primer sequences for the target genes [zonula occludens-2 (ZO2), claudin-1 (CLDN1), claudin-5 (CLDN5)] and β-actin are listed in Table 2.