Real time pcr detection system cfx 96

Total RNA was extracted from tissues or cultured cells using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. The purity and concentration of total RNA were evaluated using NanoDrop One spectrophotometer (Thermo, USA). RNA was reversely transcribed to cDNA by using the HiScript 1st Strand cDNA Synthesis Kit (Thermo, USA). Subsequent detection of RNA levels was performed with UltraSYBR Mixture kit (Vazyme, China) through Real-time PCR Detection System (CFX96, Bio-Rad, USA). For miRNA expression assay, miRNA primers were synthesized by QIAGEN and total RNA was reversely transcribed using the miScript II Reverse Transcription Kit (QIAGEN, Germany). qRT-PCR amplification for miRNA was performed by using miScript SYBR Green PCR Kit (QIAGEN, Germany). U6 were used as the internal control genes. The relative RNA expression level was calculated using the 2^−△△Ct method.

RNA was obtained using the FavorPrep Tissue Total RNA Extraction Mini Kit (Favorgen Biotech, Vienna, Austria) according to manufacturer’s instructions for animal cells. cDNA was synthesised through the iScript^™ cDNA Synthesis Kit (Bio-Rad) according to recommended protocol. TERT mRNA expression was assessed by qPCR performed with 3.5ng DNA, IQ SYBR Green Supermix (2x; Bio-Rad), and 0.5μM PCR primers (Sigma-Aldrich; S4 Table) in a Real-Time PCR Detection System CFX96 (Bio-Rad) and normalised to reference gene expression (RPS11, TBP and CPSF6, S4 Table). Data was analysed through the ΔΔCT method in Bio-Rad CFX manager software (version 3.1, Bio-Rad).

qRT-PCR was implemented using the Kit (SuperRealPreMix Plus with SYBR Green from TIANGEN, Beijing, China) on a Real-Time PCR Detection System CFX96 (Bio-Rad, Hercules, CA USA). In addition, all cDNA templates used in the experiment were of the same concentration. Twenty microliters reaction systems were preparing using with following: 1 μL of the cDNA template, 7.4 μL of water, 10 μL of 2× SuperRealPreMix Plus, 0.4 μL of 50× ROX Reference Dye, and 0.6 μL of the forward and reverse primers. The PCR program involved a two-step process that was run for 40 cycles: 95 °C for 10 min, then denaturation at 95 °C for 10 s, annealing at 60 °C for 32 s, and extension at 72 °C for 10 s. Each reaction had four replicates. Melting curve data were gathered from 65 °C to 95 °C in 0.5 °C increments. The standard curve of each primer pair was established with serial dilutions of cDNA ((1/5)⁰, (1/5)¹, (1/5)², (1/5)³, (1/5)⁴ and (1/5)⁵). The amplification efficiency (E) of qRT-PCR was determined according to the equation: E = 10^−1/K, where K represents the slope of the standard curve.

According to the prognosis of burns, tissue and blood samples were collected on days 1, 3, 7, and 14 after burns were inflicted (Hongjie et al., 2009 (link); Valvis et al., 2015 (link)). The blood was collected for serum extraction, and the wound tissue was collected for RNA analysis and fixed in 10% formalin for 24 h for HE staining. The enzyme-linked immunosorbent assay (ELISA) was performed to test the protein expression of MCP-1, IL-1 β and TNF-α in the serum. The ELISA kit was from Shanghai Jianglai Biological Technology Co., Ltd., China. The experimental procedure was conducted according to the manufacturer’s protocol. Real-time PCR was performed to test the mRNA expression of MCP-1, IL-1β, TNF-α and VEGF genes in burned tissue. The PCR reagents used were Prime Script RT Master Mix (Perfect Real Time) (RR036A, TAKARA) and TB Green Premix Ex Tap II (Tli RnaseH Plus) (RR820A, Takara, Japan). Total RNA extraction and the Real-Time PCR Detection System (CFX96, BIO-RAD, USA) was performed according to the manufacturer’s protocol. The mRNA expression of each gene in burned tissue was normalized using the average expression of β-actin protein genes and comparing that with the data obtained from the control group using the 2^−(ΔΔCT) method (Livak & Schmittgen, 2001 (link)).

Total RNA was transcribed into cDNA using with 25 mM dNTPs Mix, RT random primers, 20 U of RNase inhibitor and MultiScribe Reverse Transcriptase (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA, USA). The qRT-PCR was performed in 10-μL reactions with 5 µL of PowerUp™ SYBR™ Green Master Mix (Applied Biosystems™, Foster City, CA, USA), 50 nM for each set of primers, 2 µL of synthetized cDNA, and water to a final volume of 10 µL. All reactions were carried out using the Real-Time PCR Detection System (CFX96™; Bio-Rad Laboratories, Inc; Richmond, CA, USA). The thermal cycling profile was 50 °C for 2 min, 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. Melt curve analysis was carried out to evaluate the specificity of each PCR reaction by detection of one single peak on the dissociation curve profile. The gene relative expression levels were quantified using the (2^−ΔΔct) [33 (link)] method and GAPDH as a reference gene for cDNA normalization. Primer sequences are detailed in Table 2.

qRT-PCR was performed using the SYBR® Premix Ex Taq™ II (Perfect Real Time) (TaKaRa, Dalian, China) in a typical 20 μl PCR mixture including 10 μl of SYBR® Premix Ex Taq™ II, 1 μl of template cDNA, and 0.2 μM of each PCR primer, with double distilled water up to 20 μl. Samples were mixed gently and centrifuged briefly to collect droplets. The cycling conditions were 95°C for 5 min, followed by 40 cycles at 95°C for 20 s, 53°C for 20 s, and 72°C for 30 s, and samples were run on a Real-Time PCR Detection System CFX96 (Bio-Rad, CA, USA). All of the relative gene expression levels were calculated using the 2^−ΔΔCt method, with A. thaliana actin (AT3G18780) used as an internal control. The primers used for qRT-PCR are listed in Supplementary Table 2.