Cfx96 real time fluorescence quantitative pcr instrument

Eight pairs of CRC tissues were lysed with TRlzol Reagent (Ambion), and total RNA was isolated according to the instructions. RNA purity and concentration were tested with Nano drop. Reverse transcription of mRNA was performed using the surescript-first-strand-cDNA-synthesis-kit kit from Saville, the qRT-PCR reaction system consisted of 3 µl cDNA, 5 µl 2 × Universal Blue SYBR Green qPCR Master Mix, and 1 µl forward and reverse primers. The reaction was performed in a CFX96 real-time quantitative fluorescence PCR instrument (BIO-RAD) under the following conditions: pre-denaturation at 95 °C for 1 min, followed by 40 cycles that each involved incubation at 95 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s. The primers were synthesized by Tsingke Biotechnology, and the primer information was shown in Supplementary appendix: Supplementary Table 1. The internal control was GAPDH, three parallel experiments were set up for this experiment. Finally, the expression level of OSBPL3 between CRC tissue and para-cancerous samples was verified by qRT-PCR.

Total RNA was extracted from the tissues of infiltrative ductal carcinoma and ductal carcinoma in situ tissues using Nuclezol LS RNA Isolation Reagent Kit (ABP Biosciences Inc) following the manufacturer’s instructions.
Subsequently, cDNA was synthesized from total RNA using SureScript-First-strand-cDNA-synthesis-kit (GeneCopoeia, China). Then, RT-qPCR was performed using BlazeTaq™ SYBR ® Green qPCR Mix 2.0 (GeneCopoeia, China) on a CFX96 real-time quantitative fluorescence PCR instrument (Bio-Rad, United States) according to the following protocol: pre-denaturation at 95°C for 30s, denaturation at 95°C for 10s, annealing at 60°C for 20s, and extension at 72°C for 30s, and the whole reaction cycle 40 times.
The primers used were as follows: PLAT: fw: 5'-GCCGTGAATTTAAGGGACGC, rev: 3'-GCTCGCTGCAACCTTGGTAA, SERPINA1: fw: 5'-CCTTCACTGTCAACTTCGGG, rev: 3'-TGTGTCTCTGTCAAGCTCCT, HMGCS2: fw: 5'-TCTCTTATGGCTCTGGTTTAG, rev: 3'-GCTCTCTTTGGTTCATTATTT, MMP7: fw: 5'-TGACTCAGAAACAAAAAATGCC, rev: 3'-TACGATCCTGTAGGTGACCACT, and Gapdh: fw: 5'-CGCTGAGTACGTCGTGGAGTC, rev: 3'-GCTGATGATCTTGAGGCTGTTGTC. For each sample, qPCR assays were conducted in triplicate in a 10ml reaction volume. Using the 2−ΔΔCt method, we analyzed the RT-qPCR data by normalizing gene expression to GAPDH expression as an endogenous control.

The viral load of CV-A6, CV-A10 and CV-A16 will be detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR). The RT-PCR kit is produced by ABT Biotechnology Company (Beijing, China), the experimental operation will be carried out according to the manufacturer’s instructions. The instrument is BIO-RAD CFX96 real-time fluorescence quantitative PCR instrument.