Steponeplus rt pcr thermocycler

Total RNA was isolated from ATDC5 cells using TRizol Reagent (Thermo Fisher, Winsor, NJ, United States) and reverse transcribed according to the manufacturer’s protocol using iScript reverse transcription supermix cDNA synthesis kit (Bio-Rad, Hercules, CA, United States) 14 days after treatment. qRT-PCR was performed using the Applied Biosystems™ SYBR™ Green Assay Kit (Thermo Fisher, Winsor, NJ, United States) and Applied Biosystems StepOnePlus RT-PCR thermocycler (Applied Biosystems, San Francisco, CA, United States). Francisco, CA, United States). Primers were designed using PRIMER-Blast (NCBI) and the sequences are listed in Table 2. The evaluated mRNAs were the inflammation marker interleukin-6 (Il6), the hypertrophic chondrocyte marker type X collagen (colXa1), matrix metalloproteinase-13 (mmp13), and the chondrocyte markers type II collagen (colIIa1) and Aggrecan (acan). To evaluate the inhibitory effect of losartan on TGF-β1, mRNA expression of tgfβ1 was also evaluated. These genes of interest were normalized to the expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (gapdh) expression. Relative gene expression was calculated compared to the control culture condition (no nanofibers or losartan) using the 2^−ΔΔCT method (n = 8 per group).

Total RNA was isolated and purified from synovial fluid cells and PBMCs using TRIzol™, and transcribed into complementary DNA using the reverse transcription cDNA synthesis kit qScript™ (QuantaBio, Beverly, MA, USA, Cat#: 101414-106), according to the manufacturer’s protocol. Expression of inflammatory and senescence markers was detected by quantitative real-time PCR (qRT-PCR) using PerfeCTa^® SYBR^® Green FastMix (QuantaBio, Beverly, MA, Cat#: 101414-280) on an Applied Biosystems StepOnePlus RT-PCR thermocycler (Applied Biosystems, San Francisco, CA, USA). Primers were designed using PRIMER-Blast (NCBI), and primer sequences are listed in Table 2.

Total RNA from sorted GFP + cells was isolated using TRizol Reagent (Invitrogen, Waltham, MA, USA) and reverse transcribed using the iScript reverse transcription supermix cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. Real-time PCR (qRT-PCR) was carried out using the Applied Biosystems™ SYBR™ Green Assay kit (Thermo Fisher, Winsor, NJ, USA) and an Applied Biosystems StepOnePlus RT-PCR thermocycler (Applied Biosystems, San Francisco, CA, USA). Primers were designed using PRIMER-Blast (NCBI) and their sequence has been described in Table 1.Table 1

Primer sequences

Gene	Primer sequence (5′–3′)
GAPDH	Forward: TCCATGACAACTTTGGCATTG Reverse: TCACGCCACAGCTTTCCA
PDGFRα	Forward: TGGCATGATGGTCGATTCTA Reverse: CGCTGAGGTGGTAGAAGGAG
PDGFRβ	Forward: CCGGAACAAACACACCTTCT Reverse: TATCCATGTAGCCACCGTCA
Pax3	Forward: ACCCAAGCAGGTGACAACG Reverse: CTAGATCCGCCTCCTCCTCT
Collagen I	Forward: TCATCGTGGCTTCTCTGGTC Reverse: GACCGTTGAGTCCGTCTTTG
PPARγ	Forward: TTGCTGAACGTAAGCCCATCGAGG Reverse: GTCCTTGTAGATCTCCTGGAGCAG
Pax7	Forward: GTGCCCTCAGTGAGTTCGAT Reverse: CCACATCTGAGCCCTCATCC