Icyler iq real time pcr detection system

Total RNA was extracted from lung tissues and cells using TRIxzol Reagent (Invitrogen Corporation, CA, USA). First-strand cDNA was synthesized and amplified from 0.5 μg of total RNA using the ReverTra Ace qPCR RT Kit (Toyobo, Tokyo, Japan). Then the mRNA levels of collagen I, collagen III, TIMP-1, E-cadherin, vimentin, TGF-β1, Snail and Slug were analyzed by quantitative real-time PCR (iCyler iQ Real-time PCR Detection System, Bio-Rad Laboratories Inc., USA) using SYBR Green Real-time PCR Master Mix (Toyobo, Japan) in a total volume of 20 μL. Relative levels of mRNA expression were normalized to GAPDH expression for each gene. Primers are listed in Table 1.

The messenger RNA (mRNA) levels of the target genes were measured by quantitative real-time PCR (iCyler iQ Real-time PCR Detection System, Bio-Rad Laboratories Inc, USA) using SYBR Green Real-time PCR Master Mix (Bio-Rad) in a total volume of 20 μl. All the samples were assayed in one essay in our study. The relative quantification of gene expression was analyzed from the measured threshold cycles (C_T) by using the 2-ΔΔCt method in the experiment
[25 (link)]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard to normalize the expression level of each mRNA. Primers were designed by PRIMER 5.0. The target gene names and their primer sequences are shown in Table 
3.

The total RNA was extracted from lung tissues using TRIzol Reagent (Invitrogen Corporation, CA, United States). First-strand cDNA was synthesized and amplified from 0.5 μg of total RNA using the ReverTra Ace qPCR RT Kit (Toyobo, Tokyo, Japan). Then the mRNA levels of AT₁R were measured by quantitative real-time PCR (iCyler iQ Real-time PCR Detection System, Bio-Rad Laboratories Inc., United States) using SYBR Green Real-time PCR Master Mix (Toyobo, Japan) in a total volume of 20 μL. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard to normalize the expression level of each mRNA. Primers were designed by Sangon Biotech (Shanghai, China). The target gene names and their primer sequences were shown as follows: forward primer 5′-CCCAAGTCCACACATCAAAG-3′, reverse primer 5′-GCAAGGCAGACTGTATGGAA-3′ for AT₁R; and forward primer 5′-AAGGTGGTGAAGCAGGCGGC-3′, reverse primer 5′-GAGCAATGCCAGCCCCAGCA-3′ for GAPDH. The PCR amplification consisted of 40 cycles of denaturation (94°C, 15 s), annealing (60°C, 30 s) and extension (72°C, 30 s). All the samples were assayed in one essay in our study. The relative quantification of gene expression was analyzed from the measured threshold cycles (CT) by using the 2-ΔΔCt method in the experiment.

16HBE were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and the in vitro experiment was performed as described previously by Zhang et al. [17 (link)]. Briefly, the 16HBE cells (5 × 10³ cells per well) were seeded in a six-well plate (Corning, Corning, NY, United States) and exposed to cigarette smoke extract (CSE; 2%) for 0 h, 24 h, and 48 h. Each experiment was repeated thrice.
Total RNA was extracted from 16HBE cells, rat lung tissues, and human PBMC using TRIzol reagent and reverse-transcribed into first-strand cDNA. Real-time PCR was performed with the cDNA using SYBR Green Fast qPCR mix (Takara) with an iCyler iQ Real-time PCR Detection System (Bio-Rad Laboratories Inc., USA). The 18s was used as an internal reference, and the relative gene expression as fold change was calculated using the 2^-ΔΔCT method. All experiments were repeated three times for each gene. The target gene and their primer sequences were listed in Table S5.