Rotor gene q pcr instrument

The Real-time PCR analysis of CcTLR21 gene expression was performed with a Rotor-Gene Q PCR instrument (Qiagen) using SYBR Green Real Master Mix (Tiangen). The amplification scheme was: incubated for 1 min at 94 °C, followed by 40 cycles of 20 s at 94 °C, 20 s at 59 °C and 50 s at 70 °C. For each mRNA, gene expression was corrected by the 40S ribosomal protein S11 in each sample. Relative expression of CcTLR21 mRNA was determined using the 2^(-ΔΔCt) method. The primers used are shown in Table 1. In all cases, each PCR was performed with triplicate samples.

After cell stimulation, total RNA was extracted using an RNeasy Plus kit (catalog number 74134, Qiagen GmbH, Germantown, MD, USA), following the manufacturer’s instructions. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260/280 nm (A260/A280) using Infinite F200 Pro (Tecan Life Sciences, Männedorf, Switzerland). A total of 1 µg of RNA was reverse-transcribed using QuantiTect Reverse Transcription kit (catalog number 205313, Qiagen GmbH, Germantown, MD, USA) at 42 °C for 30 min. The reaction was stopped by incubating the tubes at 95 °C for 3 min. RT-qPCR was carried out by using QuantiNova SYBR Green PCR kit (catalog number 208056, Qiagen GmbH) in a RotorGene qPCR instrument (Qiagen GmbH, Germantown, MD, USA). The following conditions were used: 95 °C for 5 min; followed by 45 cycles of 95 °C for 20 s and 60 °C for 20 s; and then 72 °C for 20 s. The change in gene expression was quantified by the comparative Ct (cycle threshold) method (ΔCt) normalized to the expression of 60S ribosomal protein L19 (RPL19). RT-qPCR data were reported as fold increases in Ct values with respect to untreated cells. The primer sequences were: RPL19 forward 5′-GAT GCC GGA AAA ACA CCT TG-3′. RPL19 reverse: 5′-TGG CTG TAC CCT TCC GCT T-3′. TGF-β1 forward: 5′-AGCAGCACGTGGAGCTGT-3′. TGF-β1 reverse: 5′-CAGCCGGTTGCTGAGGTA-3′. All primers were from Merck KGaA.

Total RNA was extracted from various tissues and EPC cells using an TRIzol reagent (TIANGEN). First-strand cDNA was synthesized using FastQuant RT kit (TIANGEN) in accordance with the manusfacture’s instructions. Real-time PCR was performed in a Rotor-Gene Q PCR instrument (Qiagen) with TransStart Tip Green qPCR SuperMix (Transgen). Real-time PCR conditions were 94 °C for 30 s, followed by 94 °C for 5 s, 60 °C for 30 s, and 70 °C 50 s for 40 cycles. Reactions were performed in 20 μl volume containing 10 μl SYBR green real-time PCR master mix, 6.8 μl double-distilled water, 0.6 μl of each primer, and 20 ng (2 μl) cDNA template. All samples were analyzed in triplicates and the expression value of all genes in common carp was calculated as relative to 40S ribosomal protein S11 gene or β-actin of EPC cells with the 2^{(−∆∆C(T))} method [42 (link)]. The primers were listed in Table 1.