Myiq2 sequence detection system

DRGs were subjected to TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for RNA extraction. The purity and concentration of the extracted RNA samples were determined by spectrophotometric analysis. Reverse transcription was performed using an EasyScript First-Strand cDNA Synthesis Super Mix kit (TransGen Biotech, Beijing, China) with 3 micrograms of RNA. Quantitative real-time PCR (qRT-PCR) was used to evaluate the relative mRNA expression levels of mouse Atf3, Sox11, Lin28a, Gap43, Smad1, Lin28b, Jun and Gapdh (Invitrogen), utilizing TransStart Eco Green qPCR Super Mix (TransGen Biotech). The Bio-Rad myiQ2 Sequence Detection System (Bio-Rad, Hercules, CA, USA) was employed in this study. Primers purchased from Invitrogen were utilized as per Supplementary Table S1 instructions while cycle conditions followed the specifications of the primers. Relative gene expression levels were normalized against Gapdh and β-actin and analyzed using the 2^−ΔΔct method.

Total RNA was extracted from human mesenchymal stem cells (h-MSCs) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration and purity of RNA samples were assessed by spectrophotometric analysis. Three micrograms of RNA was used to reverse transcription by using an EasyScript First-Strand cDNA Synthesis Super Mix kit (TransGen Biotech, Beijing, China). The relative mRNA expression of human P2X7, RUNX2, ALP, OPN, and GAPDH (Invitrogen) was evaluated by quantitative real-time PCR (qRT-PCR) by using the BioRad myiQ2 Sequence Detection System (BioRad, Hercules, CA, USA) and TransStart Eco Green qPCR Super Mix (TransGen Biotech, Beijing, China). Primers were purchased from Invitrogen, and the sequences were listed in Additional file 1: Table S1. Cycle conditions followed primers’ introduction. Relative gene expressions were normalized with GAPDH and analyzed by the 2^−ΔΔCt method.