Blazetaq sybr green qpcr mix 2

Total RNA of ten DM-ILD patients and ten control subjects were isolated from peripheral blood using TRIzol reagent (15596018, Life Technologies, American). And RNA was reverse-transcribed to cDNA via the SureScript-First Strand cDNA Synthesis Kit (QP057, GeneCopoeia, Guangzhou, China). The expression of PLAUR was detected by Bio-Rad CFX96 (American) using BlazeTaq™ SYBR^® Green qPCR Mix 2.0 (QP033, GeneCopoeia, Guangzhou, China). PCR amplification cycle conditions were 95°C 30 s, 95°C 10 s, 60°C 20 s, and 72°C 30 s for 40 cycles. GAPDH was used as the internal standardized reference. The relative expression level was calculated using the 2^−ΔΔCt method. The specific primers were as follows: PLAUR, 5′-CGAGGTTGT GTGTGGGTTA-3’ (forward) and 5′-GGC​ACT​GTT​CTT​CAG​GGC​T-3’ (reverse); GAPDH, 5′- CGC​TGA​GTA​CGT​CGT​GGA​GTC-3′ (forward) and 5′- GCTGATGAT CTTGAGGCTGTTGTC-3′ (reverse).

Total RNA was extracted from mouse eyes and cell lines using an RNAfast 200 total RNA rapid extraction kit (Feijie Biotechnology, Shanghai, China). After the quantity and purity were determined by NanoDrop 2000 (Thermo, Shanghai, China), 1 μg of total RNA was used to generate cDNA by miDETECT A Track miRNA qRT-PCR Starter Kit (RiboBio, Guangzhou, China). Quantitative RT-PCR analysis was performed using Blaze Taq™ SYBR Green qPCR Mix 2.0 (GeneCopoeia, Guangzhou, China) to detect the mRNA levels of interleukin (IL)-1β, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-α. Primers used for qRT-PCR are listed in Table 1. The relative mRNA expression of each target gene was normalized to that of the housekeeping gene β-actin and GAPDH, and the result was fold-changed compared to the blank control group (set to 1).

The mRNA expression levels of prognostic genes were detected in 10 pairs of ccRCC tissue samples and para-cancerous control tissue samples from the First Hospital of ShanXi Medical University. This study was allowed by the Ethics Committee of the First Hospital of ShanXi Medical University. All patients had approved for the use of clinical tissues for research purposes. Total RNA was isolated using TRIzol (Genecopoeia). The SureScript-First-strand-cDNA-synthesis-kit (Genecopoeia) was used for first-strand cDNA synthesis. For the analysis of the target gene mRNA levels, qPCR was performed using BlazeTaq™ SYBR ^® Green qPCR Mix 2.0 according to the manufacturer’s instructions (Genecopoeia). The relative expression of mRNA was calculated by the 2^−ΔΔCt method with the normalization to GAPDH. All specific primers were shown in Table 1.

Total RNA from freshly isolated PBMCs (section 2.7) or cell cultures was extracted with TRIzol Reagent (Invitrogen, San Diego, CA, USA). The cDNA was synthesized using a SureScript™ First-Strand cDNA Synthesis Kit (GeneCopoeia, Guangzhou, China). The mRNA levels were determined by RT-qPCR with BlazeTaq™ SYBR^® Green qPCR Mix 2.0 (GeneCopoeia) and GAPDH as the internal control. The primers are listed in Supplementary Table S2. Total RNA was reverse-transcribed and quantified with an All-in-One™ miRNA RT-qPCR Detection kit (QP015, GeneCopoeia). U6 small nuclear RNA was the internal control.

Total RNA was extracted from cells by TRIzol and then reverse transcribed into cDNA by RevertAid First Strand cDNA Synthesis Kits (Thermo Fisher Scientific, MA, United States). qPCR was performed using Blaze Taq™SYBR®Green qPCR Mix 2.0 (GeneCopoeia, Maryland, United States). The target gene expression was normalized according to the internal reference gene β-actin. The primers used in this study are as follows: DDIT4 forward, 5′-GGTTTGACCGCTCCACGAG-3′ and reverse, 5′-ATCCAGGTAAGCCGTGTCTTC-3′; β-actin forward, 5′-CTACCTCATGAAGATCCTCACCGA-3′ and reverse, 5′-TTCTCCTTAATGTCACGCACGATT-3′; Rictor forward, 5′-TTCCCTTTCTTTGCTTCT-3′ and reverse, 5′-GTGTTCTGATTCGCCTGT-3′; Raptor forward, 5′-CGTAGCCGACAAGGACAGCA-3′ and reverse, 5′-CGTCAGCAGAAGCGAGCAGT-3′.

Total RNA from 8 normal endometrium samples, 8 EU samples and 8 EC samples was isolated using Nuclezol LS RNA Isolation Reagent following the manufacturer’s instructions (ABP Biosciences Inc.). Next, total RNA was reverse transcribed into cDNA utilizing the SureScript-First-strand-cDNA-synthesis-kit (GeneCopoeia), according to the manufacturer’s protocol. qPCR was subsequently performed using the BlazeTaq™ SYBR ^® Green qPCR Mix 2.0 (GeneCopoeia). The following thermocycling conditions were used for qPCR: 1 cycle at 95°C for 30 sec (initial denaturation), followed by 40 cycles of 10 sec at 95°C (denaturation), 20 sec at 60°C (annealing), and 30 sec at 72°C (extension). The sequences of the primers were listed in Table 1. The relative expression level was normalized to the endogenous control GAPDH and calculated using the 2^−ΔΔCq method (27 (link)). The student’s t-test was used to compare the differences between the two groups. The two-tailed P-value < 0.05 in statistical analysis was defined as statistically significant.