Sybr green 1 pcr master mix

Quantification of caspase-4 expression in 293T cells was performed with SYBR Green I PCR Master Mix (Takara) in the StepOne Plus Real-Time PCR System (ABI). The primers used for each gene examined are listed below. CASP4: 5′-CAGACTCTATGCAAGAGAAGCAACGTATGGCAGGA-3′ (forward) and 5′-CACCTCTGCAGGCCTGGACAATGATGAC-3′ (reverse); actin: 5′-CATGTACGTTGCTATCCAGGC-3′ (forward) and 5′-CTCCTTAATGTCACGCACGAT-3′ (reverse).

Cells were washed with cold PBS, and total RNA was extracted from chondrocytes (cells) and joint residue (tissue) with the RNeasyPlus Mini Kit (Qiagen, Shanghai, China) with a genomic DNA eliminator according to the manufacturer's instructions. The extracted RNA samples were dissolved in RNase‐free water and quantified by a spectrophotometer instrument measuring absorbance at 260 nm. After treatment with DNase I, RNA (0.5‐2 μg) was reverse‐transcribed to cDNA with the first‐strand cDNA synthesis kit (Mbi, Glen Burnie, MD, USA).
The sets of primers used to detect each gene in the TGF‐β super family are listed in Table 1. The BLAST programme was used to determine all primer sequences, and all samples were normalized to the housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Quantitative real‐time PCR (qPCR) was performed by amplifying a specific product with a reaction mixture containing cDNA, SYBR Green I PCR master mix (Takara, Tokyo, Japan) and primer pairs, and qPCR was run on an ABI 7300 instrument (Applied Biosystems, Shanghai, China) equipped with 96‐well optical reaction plates. The reaction conditions are as follows: 95°C for 10 minutes followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Each gene was assayed in triplicate. The fold change calculations were performed with the 2−ΔΔCt method.

Before being subjected to RNA extraction, macrophages were incubated in medium containing 30% CM from TSP50-o/e cells or control cells for 24 hours. Total RNA was extracted according to the manufacturers’ instructions (Invitrogen). 50 ng of cDNA was subjected to real-time PCR in a final volume of 10 μL, containing one set of primers and the SYBR Green I PCR master mix (TAKARA, Dalian, China). The amplification was carried out with an ABI thermocycler (Applied Biosystems) as follows: 95°C for 1 min and then 40 cycles of 95°C for 5s and 60°C for 1min. Relative expression of each gene was calculated using the ΔΔCT method. The data was expressed as the relative expression of the target genes between the experimental group and the control group. All real-time PCR reactions were performed in triplicate.

Total RNA was extracted from skin samples stored in liquid nitrogen using TRIzol (Invitrogen, Carlsbad, CA, USA), and then resuspended in RNase-free water. The RNA was electrophoresed in 1% agarose gels to confirm its quality and quantity. Equal amounts of RNA (1 µg) were reverse-transcribed using a cDNA synthesis kit (Toyobo CO. LTD., Japan).
qPCR was performed on a CFX 96™ Real-Time PCR detection system (Bio-Rad mini option, USA) using SYBR Green I PCR Master Mix (Takara BIOINC., Japan). The following oligonucleotides were used as primers: the internal control β-actin (F:5′-GAGTACAACCTTCTTGCAGCTC-3′ and R: 5′-CATACCCACCATCACACCCTG-3′), rat collagen I-α₁ (F: 5′-GATGGACTCAACGGTCTCCC-3′ and R: 5′-CGGCCACCATCTTGAGACTT-3′), rat collagen III (F: 5′-CTGAAGGGCAGGGAACAACT-3′, R: 5′-ATCCCGAGTCGCAGACACATA-3′), and rat MMP-3 (F: 5′-GATGGACTCAACGGTCTCCC-3′, R: 5′-CGGCCACCATCTTGAGACTT-3′). The two-step qPCR amplification standard procedure was as follows: Step 1, 95°C, 30 s; Step 2, 40 cycles with 95°C, 5 s and 60°C, 30 s. The quantity of PCR products was calculated from the cycle threshold value. The levels of gene expression were normalized with those of β-actin gene. The data was calculated with the formula of relative expression quantity = 2^−△△Ct.