Sybr green qpcr super mix udg with rox

Total RNA was extracted using TRIzol (Life Technology, Carlsbad, CA, USA) according to the manufacturer's instructions. Real-time PCR amplification and detection were performed using the SYBR Green qPCR SuperMix-UDG with ROX (Life Technologies) in a fluorescence thermal cycler (StepOne Real-Time PCR system, Life Technologies) according to the manufacturer's protocol. Gene expression was normalized using GAPDH as a reference gene. Relative mRNA expression levels were calculated following the ΔΔCt method with the following primers: GAPDH, IL-1β, TNF-α, IL-32β, IL-32γ, CLS1, and NLRP3 in Table 1. All amplifications were conducted within the linear range of the assay and normalized to respective GAPDH levels using SPSS Version 18.0 (SPSS Institute, Inc., Chicago, IL, USA).

Total RNA was extracted using TRIzol (Life Technologies, Carlsbad, USA) according to the manufacturer's instructions. Real-time polymerase chain reaction (PCR) amplification and detection were performed using the SYBR Green qPCR SuperMix-UDG with ROX (Life Technologies) in a fluorescence thermal cycler (StepOne real-time PCR system, Life Technologies) according to the manufacturer's protocol. Gene expression was normalized using GAPDH as a reference gene. The primers used in our study are as follows: RORα, forward: 5′-CAG GCT TCT TTC CCT ACT GTT CGT-3′, reverse: 5′-CCG CTG CTT GTT TTG ATA GTT CTC-3′; BMAL1, forward: 5′-CAA TCC ATA CAC AGA AGC AAA CTA C-3′, reverse: 5′-ACA TCC TAC GAC AAA CAA AAA TCC-3′; P16-INK4A (CDKN2A), forward: 5′-TTT TCA CTG TGT TGG AGT TTT CTG G-3′, reverse: 5′-TGA GCT TTG GTT CTG CCA TTT G-3′; IL-1α, forward: 5′-GCC CAA GAT GAA GAC CAA CCA GT-3′, reverse 5′-CCG TGA GTT TCC CAG AAG AAG AGG-3′; GAPDH, forward: 5′-CGC TGA GTA CGT CGT GGA GTC-3′, reverse: 5′-GCT GAT GAT CTT GAG GCT GTT GTC-3′; 18S rRNA, forward: 5′-AGG TCT GTG ATG CCC TTA GAT GTC-3′, reverse: 5′-TCC TCG TTC ATG GGG AAT AAT T-3′.

Total RNA was extracted using TRIzol (Life Technologies) according to the manufacturer’s instructions. Real-time polymerase chain reaction (PCR) amplification and detection were performed using the SYBR Green qPCR SuperMix-UDG with ROX (Life Technologies) in a fluorescence thermal cycler (StepOne Real-Time PCR system, Life Technologies) according to the manufacturer’s protocol. Gene expression was normalized by using GAPDH as a reference gene. The following primers were used in our study: human EPO forward: 5′-TAT-GCC-TGG-AAG-AGG-ATG-GA-3′, reverse: 5′-AGA-GCC-CGA-AGC-AGA-GTG- GT-3′; rat EPO forward: 5′-AGA-ATG-AAG-GTG-GAA-GAA-CAG-G-3′, reverse: 5′-CCG- AAG-CAG-TGA-AGT-GAG-G-3′; human GAPDH, forward: 5′-GAT-CCC-GCT-AAC-ATC- AAA-TG-3′, reverse: 5′-GAG-GGA-GTT-GTC-ATA-TTT-CTC-3′; rat GAPDH, forward: 5′-AGA-CAG-CCG- CAT-CTT-CTT-GT-3′, reverse: 5′-TAC-TCA-GCA-CCA-GCA-TCA-CC-3′. The primers were synthesized by Shanghai Generay Biotech Co., Ltd (Shanghai, China).

Total RNA was isolated with RNeasy Min Elute clean-up kit (Qiagen, ref. 74204), according to the manufacturer’s protocol. Concentration and RNA quality were evaluated by NanoDrop (ND-1000 Spectrophotometer; Thermofisher Scientific). 600 ng of RNA was reverse-transcribed (RT) to generate cDNA using iScript reverse transcription kit (Biorad, ref. 170–8841), following manufacturer’s instruction. 2, 7 ng of cDNA were amplified by quantitative PCR (qPCR) using SYBR Green qPCR SuperMix-UDG with Rox (Thermofisher, ref. 11733046) and 0.1 µM of forward and reverse primers. Levels of transcripts (Ct) were normalized to those of the housekeeping gene GAPDH (ΔCt) and subsequently to the mean ΔCt of the reference sample (ΔΔCt). Results were reported as relative quantities (RQ = 2^−ΔΔCt). The sequences of forward and reverse primers used for qPCR analyses are listed in Supplementary Table 6.

Thioglycollate-elicited peritoneal macrophages were isolated as described above. Macrophages were washed with DMEM supplemented with 10% FBS, 2 mM l-glutamine, 50 µg/mL penicillin and 50 U/mL streptomycin. Neutrophils were elicited and isolated as described above. Isolated neutrophils were centrifuged at 500× g, resuspended in PBS containing 90% Percoll and purified by ultracentrifugation using a Type 90 Ti Rotor (Beckman Coulter, Mississauga, ON, Canada) as described by others [61 (link)]. Total RNA from macrophages and neutrophils were extracted using the RNeasy mini kit (Qiagen, Toronto, ON, Canada) and RNA integrity and concentration were evaluated by measuring the absorbance at 260/280 in a spectrophotometer. In addition, 1 µg of RNA was used to prepare cDNA using the Quantitec reverse transcription kit (Qiagen, Toronto, ON, Canada). Quantitative real-time-(RT-)PCR was performed on prepared cDNA using a mixture of SYBR Green qPCR SuperMix-UDG with ROX (Thermo Fisher Scientific, Ottawa, ON, Canada) and primers listed in Table 1. Quantitative real-time-(RT-)PCR was run on an Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Ottawa, ON, Canada).

Total RNA extracted (500–800 ng per sample) from articular cartilage was reverse-transcribed with RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Milan, Italy) in a 20 μL reaction solution. Quantitative RT-PCR was performed using one-twentieth of the RT products and platinum SYBR Green qPCR SuperMix UDG with Rox (Invitrogen Life Technologies, Milan, Italy). The primers used are shown in Table 2. The reaction was followed by a melting curve protocol according to the specifications of the ABI 7900 instrument (Applied Biosystems, Foster City, CA, USA). Rat β-Actin (ACTB) was used as a housekeeping gene for normalization. Data are presented as mean ± SD of at least three independent experiments. Differences were analyzed by Student’s t test, with p < 0.01 being considered statistically significant.