Pcr master mix kit

Total RNA from NP cells was extracted using an RNA purification kit (Sangon, RN001) following the manufacturer’s protocols. A PCR master mix kit (Toyobo, 0910–012700) was used to remove the genomic DNA and to reverse-transcribe RNA into complementary DNA (cDNA). Custom-designed primers targeting mRNA encoding IL-6, type II collagen, and visfatin were synthesized for qPCR. PCR using primers and cDNA templates was performed with SYBR Green Mix (Roche, 47,138,600). Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. The relative mRNA expression levels were quantified using the 2-ΔΔCt method. All RT-qPCR experiments were carried out in triplicates. The primer sequences used are listed in (Table 2).Table 2.

Primer sequences (human)

Gene	Forward prime (5ʹ-3ʹ)	Reverse prime (5ʹ-3ʹ)
IL-6	ACTCACCTCTTCAGAACGAATTG	CCATCTTTGGAAGGTTCAGGTTG
Visfatin	GTGACTTAAGCAACGGAGCG	GGAGGATGTTGAACTCGGCT
Aggrecan	GCACAGCCACCACCTACAAAC	CTTTGGCATTCGGCGGACAA
Collagen II	ATGAGGGCGCGGTAGAGAC	TCACAGACACAGATCCGGCA
MMP3	GGTTCCGCCTGTCTCAAGAT	AGGGATTTGCGCCAAAAGTG
ERK1/2	TACACCAACCTCTCGTACATCG	CATGTCTGAAGCGCAGTAAGATT
JNK	TGTGTGGAATCAAGCACCTTC	AGGCGTCATCATAAAACTCGTTC
p38	CCAGGGGCTGAGCTTTTGAA	TCGGCCACTGGTTCATCATC
GAPDH	GACAGTCAGCCGCATCTTCTT	AATCCGTTGACTCCGACCTTC

Total RNA was extracted from PBMCs using Trizol RNA extraction reagent (Life Technologies, Carlsbad CA, USA) and reverse-transcribed into cDNA (PCR MasterMix kit, Toyobo Co., Ltd., Osaka, Japan). Primers of each gene were designed using Primer Premier 5.0 (PREMIER Biosoft, Palo Alto, CA, USA) according to the cDNA sequences obtained from the National Center for Biotechnology Information. The primer sequences and amplicon sizes are shown in Table 1. Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green Premix Ex Taq in a Mastercycler ep realplex instrument (Eppendorf AG., Hamburg, Germany). The PCR protocol was as follows: predenaturation for 20 min at 95°C followed by denaturation for 30 s at 95°C, annealing for 30 s at 54-55°C, and extension for 40 s at 72°C for 40 cycles. The relative amount of target gene mRNA was normalized to GAPDH.