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).
Iscript reverse transcription supermix cdna synthesis kit
Manufactured by Bio-Rad
Sourced in United States
The IScript reverse transcription supermix cDNA synthesis kit is a reagent used for the conversion of RNA into complementary DNA (cDNA). The kit contains a reverse transcriptase enzyme, buffer, and other necessary components for the reverse transcription reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Steponeplus rt pcr thermocycler, by Thermo Fisher Scientific (2 mentions)
Sybr green assay kit, by Thermo Fisher Scientific (2 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Stepone plus real time pcr thermocycler, by Thermo Fisher Scientific (1 mentions)
5 protocols using iscript reverse transcription supermix cdna synthesis kit
1
Quantification of Chondrocyte Gene Expression
2
Gene Expression Profiling by qRT-PCR
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Total RNA samples were used for cDNA synthesis with iScript reverse transcription supermix cDNA Synthesis Kit (Bio-RAD®) according to manufacturer instructions. Once cDNA was obtained, real-time PCR was developed for IL1B, IL-6, IL10, TGFβ, TNFα, and GAPDH genes. The primer used for IL1β was F: 5′-AAGCTGATGGCCCTAAACAG-3′, R: 5′-AGGTGCATCGTGCACATAAG-3′; IL6 was F: 5′-CCAGCTATGAACTCCTTCTC-3′, R: 5′-GCTTGTTCCTCACATCTCTC-3′; IL10 was F: 5′-TCTCCGAGATGCCTTCAGCAGA-3′, R: 5′-TCAGACAAGGCTTGGCAACCCA-3′; TGFβ was F: 5′-TACCTGAACCCGTGTTGCTCTC-3′, R: 5′-GTTGCTGAGGTATCGCCAGGAA-3′; TNFα was F: 5′-CTCTTCTGCCTGCTGCACTTTG-3′, R: 5′-ATGGGCTACAGGCTTGTCACTC-3′; and GAPDH was F: 5′-ACCCACTCCTCCACCTTTGA-3′, R: 5′-CTGTTGCTGTAGCCAAATTCGT-3′.
3
BAFF-R mRNA Expression Quantification
4
Chondrogenic Differentiation of MDSCs
5
Gene Expression Analysis of Cell Subpopulations
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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 .
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 |
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