Cfx connect read time pcr detection system

Total RNA was subjected to reverse transcription with ReverTra Ace qPCR RT Master mix according to the manufacturer's instructions (Toyobo). The DNA samples were amplified with a CFX Connect Read-Time PCR Detection System (BioRad), THUNDERBIRD SYBR qPCR Mix (Toyobo), and the primer sets specific for human genes. The sequences of primer sets were as follows: forward primer, 5ʹ-CTGGAGTACTATGAGCGGGC-3ʹ and reverse primer, 5ʹ-TGGCTGATATCTGGGTGCCT-3ʹ for IFIT1; forward primer, 5ʹ-TGAGGAAGGGTGGACACAAC-3ʹ and reverse primer, 5ʹ-ACATCGCAATTGCCAGTCCA-3ʹ for IFIT3; forward primer, 5ʹ-CTGGATAGCAGCAGCCTCAG-3ʹ and reverse primer, 5ʹ-AGCTCCATAAGGAAGCACTCG-3ʹ for IRF7; forward primer, 5ʹ-TGGCATAACCAGAGTGGCTG-3ʹ and reverse primer, 5ʹ-CACCACCAGGCTGATTGTCT-3ʹ for Mx1; forward primer, 5ʹ-ACCATGCACTCTGTTTGCGA-3ʹ and reverse primer, 5ʹ-CGAAAGGCACCTATCCGTTC-3ʹ for TLR3; forward primer, 5ʹ-GCACCAACTACCCAGTGGAG-3ʹ and reverse primer, 5ʹ-TGGCGTCTGGTCTTTGACAG-3ʹ for TICAM1; forward primer, 5ʹ-AAGGCTGGGGCTCATTTGCA-3ʹ and reverse primer, 5ʹ-ATGACCTTGCCCACAGCCTT-3ʹ for GAPDH.
Each mRNA expression level was normalized with respect to the mRNA expression of GAPDH. Data are presented as means ± S.D. (n = 4).

Total mRNA was isolated from the purified lung or skeletal muscle ECs using the RNeasy Mini Kit (QIAGEN) and reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad), according to the manufacturer's instructions. Real time reverse transcriptase qPCR (RT-qPCR) was analyzed on the CFX Connect Read-Time PCR Detection system (Bio-Rad) via SYBR Green (Bio-Rad). Gene expression levels were normalized to the housekeeping gene Gapdh or β-actin. The relative gene expression level of each sample was compared with an internal control. Primers used are listed in Supplementary Table 1.