Abi prism sds 7300 system

RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, United States) after transduction. RevertAid First Strand cDNA Synthesis kit (#K1622, Fermentas, Waltham, MA, United States) was used for first-strand cDNA synthesis. Quantification of mRNA USP7 levels was conducted using SYBR Green polymerase chain reaction (PCR) Mix (Thermo Fisher, Shanghai, China) on ABI Prism 7300 SDS System (Applied Biosystem, Foster City, CA, United States). GAPDH was used as internal control. The primer sequences used in this study were USP7-forward, 5′-GGG​CGT​GAA​GTT​CCT​GAC​C-3′; USP7-reverse, 5′-CAT​TTG​CCA​TCC​CCT​TTG​G-3′; NOX4-forward, 5′-TGC​CCA​CTT​GGT​GAA​CGC-3′; NOX4-reverse, 5′-TCA​ACA​AGC​CAC​CCG​AAA​C-3′; GAPDH-forward, 5′-GGA​GTC​TAC​TGG​CGT​CTT​CAC-3′; and GAPDH-reverse, 5′-ATG​AGC​CCT​TCC​ACG​ATG​C-3′.

Total RNA was isolated from the HUVECs using the RNeasy Mini Kit (Qiagen, Valencia, CA) and the quantity was measured using a spectrophotometer, as previously described. Total RNA was transcribed using the PrimeScript RT Reagent Kit with the gDNA Eraser (TaKaRa, Dalian, China). The primers that were used for qPCR are listed in Table 1 (Sangon Biotech, Shanghai, China). The cDNA was assessed using Real-Time PCR with SYBR Premix Ex Taq (TaKaRa, Dalian, China). Thermal cycling was performed using an ABI Prism SDS 7300 system (Applied Biosystems). Gene expression was compared according to C_t values, as previously described.

Human Inflammasomes PCR Array was performed to evaluate the expression of 84 inflammasomes-related genes. Total RNA was extracted from PBMCs using the RNeasy Mini Kit. An UNIC 2800 UV/VIS Spectrophotometer was used to assess the quantity and quality of the RNA extracts by measuring the absorbance at 260 and 280 nm. Total RNA was purified using an RNase-Free DNase Set. cDNA was generated by reverse transcription of 20 ng of total RNA from each sample using the RT² First Strand Kit and then combined with the RT² SYBR® Green ROX qPCR Mastermix in 96 well arrays. Thermal cycling was performed using an ABI Prism SDS 7300 system (Applied Biosystems). Gene expression was compared using the Ct values and calculated using the ΔΔCt method with normalization to the average expression levels of five common genes (ACTB, B2 M, GAPDH, HPRT, and RPL13A) [16 (link)].

The Human Oxidative Stress PCR Array was used to evaluate the relative expression of 84 oxidative stress-related genes. Total RNA was isolated from the HUVECs using the RNeasy Mini Kit (Qiagen, Valencia, CA). UNIC 2800 UV/VIS Spectrophotometer was used to assess the quantity and quality of the RNA extracts by measuring the absorbance at 260 and 280 nm. Total RNA was purified using RNase-Free DNase Set (Qiagen, Valencia, CA). cDNA was generated by reverse transcription of 20 ng of total RNA from each sample using the RT² First Strand Kit (Qiagen, Valencia, CA) and was then combined with the RT² SYBR Green ROX qPCR Mastermix in 96-well arrays. Thermal cycling was performed using an ABI Prism SDS 7300 system (Applied Biosystems). Gene expression was compared using C_t values and the results were calculated using ^ΔΔC_t method with normalization to the average expression levels of five common genes (ACTB, B2M, GAPDH, HPRT, and RPL13A) [10 (link)].

The expression of 84 inflammation-related genes was evaluated using quantitative real-time PCR array (SABiosciences, Valencia, CA) according to the instructions. Total RNA was isolated using the RNeasy Mini Kit and then was quantified and qualitied by measuring the absorbance at 260 and 280 nm. Total RNA was purified with DNase. cDNA synthesis was performed by reverse transcription of 20 ng total RNA as described for RT-PCR and then combined with the SYBR Green Master Mix in 96-well plates following the manufacturer's recommendations. Thermal cycling was performed using an ABI Prism SDS 7300 system (Applied Biosystems, Madrid, Spain).

Gene expression analysis of 84 inflammation-related genes was performed using quantitative real-time PCR (qPCR) arrays based on the manufacturer’s instructions. Total RNA was isolated using an RNeasy Mini Kit, and the concentration of the extracted RNA was then quantified by measuring the absorbance at 260 and 280 nm. In addition, cDNA synthesis was performed by a Reverse Transcription Kit of 20 ng total RNA, then combined with SYBR Green Master Mix (Qiagen, New York, NY, USA) in 96-well plates following the manufacturer’s instructions. A Human Inflammasomes PCR Array, an RNeasy Mini Kit, an RNase-free DNase Set, a Reverse Transcription Kit, and SYBR Premix Ex Taq II were all obtained from Qiagen (New York, NY, USA). Thermal cycling was performed using an ABI Prism SDS 7300 system (Applied Biosystems, Waltham, MA, USA).