Bestar sybr green rt pcr master mix

To elucidate the validity of the RNA-seq data, qRT-PCR experiments were performed for some DEGs. The relative gene expression of each gene was calculated using the Livak and Schmittgen 2^−ΔΔCt method^{34 (link)}, normalized with the GAPDH gene of rat. The same RNA samples for RNA-seq were used for qPCR. In each sample, l μg of pooled RNA was reversely transcribed using the PrimeScriptTM RT Reagent Kit (Takara, Dalian, China) according to the instruction from the manufacturer. qPCR was performed on the Bio-Rad S1000 with Bestar SYBR Green RT-PCR Master Mix (DBI Bioscience, Shanghai, China). The PCR conditions are consisted of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min. Primer sequences: C3: 5′-GCG GAA GTG TTG TGA GGA TG-3′ (forward), 5′-ATG GTC TCT TCT GTG CTG CT-3′ (reverse); Cd53: 5′-CAC TGA ACT GCC AGA TTG ACA-3′ (forward), 5′-CCT TAT GGA ATG GGT GCT TTG A-3′ (reverse); Cxcl1: 5′-CGA TGG TCG TTC AAT TCC AAT T-3′ (forward), 5′-ATC TCT CCG CCC TTC TTC C-3′ (reverse); Il1b: 5′-TTT CCC TCC CTG CCT CTG A-3′ (forward), 5′-GAC AAT GCT GCC TCG TGA C-3′ (reverse); GAPDH: 5′-AAG TTC AAC GGC ACA GTC AAG-3′ (forward), 5′-ACA TAC TCA GCA CCA GCA TCA-3′ (reverse).

In this study, to elucidate the validity of the RNA-seq data, quantitative real-time PCR (qPCR) was performed for some selected DEGs, and normalized with the reference gene GAPDH gene of sheep. The information of primers is presented in Table 1. The same RNA samples for RNA-seq were used for qPCR. In each pooled sample, l μg of RNA was reversely transcribed using the PrimeScript^TM RT Reagent Kit (Takara, Dalian, China) following the manufacturer’s instructions. qPCR was performed on the Bio-Rad S1000 with Bestar SYBR Green RT-PCR Master Mix (DBI Bioscience, Shanghai, China). The PCR conditions are consisted of denaturing at 95°C for 10 min, 40 cycles of denaturing at 95°C for 15 s, annealing and extension at 60°C for 1 min. PCR amplifications were performed in triplicate for each sample.

The total RNA of HepG2 cells transfected with different vectors was isolated using TRIzol reagent (Ambion, Austin, TX, USA). Then, cDNA was synthesized. qPCR was performed using Bestar SYBR Green RT-PCR Master Mix (DBI Bioscience, Shanghai, China) on the Bio-Rad S1000. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a reference gene for assessing the efficiency of PAIP1 knockdown. The primers used for qPCR are listed in Table S1. The expression of PAIP1 was then normalized to GAPDH using 2^−ΔΔCT.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. We synthesized the cDNA using standard methods, and we performed the RT-qPCR analysis on the Bio-Rad S1000 using the Bestar SYBR Green RT-PCR Master Mix (DBI Bioscience, Shanghai, China). Detailed primer sequences are listed in Table 1. Using GAPDH as a normalization factor, transcript expression was normalized and calculated by the 2-ΔΔCT method (Livak & Schmittgen, 2001 (link)). Statistical comparisons were made using paired Student’s t test in GraphPad Prism 7.0 software (Graphpad Prism software; GraphPad, San Diego, CA, USA).

The cDNA synthesis was done by standard procedures and real time PCR was performed on the Bio-Rad S1000 with Bestar SYBR Green RT-PCR Master Mix (DBI Bioscience). The concentration of each transcript was then normalized to GAPDH mRNA level using the 2^−ΔΔCT method [66 (link)]. Comparisons were performed with the paired Student’s t-test.
In brief, for the preparation of total cell lysates, the HeLa cells were lysed in RIPA buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1.0% deoxycholate, 1% Triton X-100, 1 mM EDTA and 0.1% SDS. The samples were centrifuged (12,000 rpm, 5 min) and the supernatants were further analyzed on a 10% SDS-PAGE gel and subsequently transferred to a PVDF membrane (Millipore). HMGB1 was detected using monoclonal Flag antibody (Sigma) and HMGB1 antibody (ab79823, Abcam) diluted in TBST (1:2,000), and Action (Abclonal) was used as a loading control (1:2,000).

GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a control. cDNA synthesis was performed using standard procedures and real time PCR was performed on the Bio-Rad S1000 with Bestar SYBR Green RT-PCR Master Mix (DBI Bioscience). The concentration of each transcript was then normalized to the level of GAPDH mRNA using the 2- ΔΔCT method [60 (link)]. Comparisons were performed with a paired Student’s t-test by using GraphPad Prism software (San Diego, CA).