Stepone plus thermal cycler and detection system

Cells were seeded at 2 × 10⁵ cells/ml and cultured in the presence of microparticles (with and without IGF-1) for 5, 7, and 10 days. Total RNA was isolated using RNeasy Mini Kit (Qiagen, USA) following manufacturer’s instructions. The purity and concentration of total cellular RNA was determined using Nanodrop-1000, version 3.6.0. The minimum RNA amount obtained was considered as a base, for calculations to prepare reverse transcribe complementary DNA (cDNA) using the Verso cDNA kit (Thermo Scientific, USA) according to manufacturer’s protocols. The forward and reverse primers for the selected genes were designed from Integrated DNA Technologies (IDT, USA) and are listed in Table 1. Expression was quantified using real time RT-PCR analysis with SYBR green master mix kit (Applied Biosystems, USA). Data analysis was carried out using Applied Biosystems StepOne Plus thermal cycler and detection system. The real time RT-PCR analysis was carried out for two independent experiments with each sample run in duplicates. The gene expression levels were normalized to the expression of the housekeeping gene GAPDH and were expressed as fold changes relative to the expression of the genes in cells only on the respective days.

Cells were seeded at 2 × 10⁵ cells/ml and cultured in the presence of microparticles (with and without BMP-7) for 5, 7 and 14 days. Total RNA was isolated using RNeasy Mini Kit (Qiagen, USA) following manufacturer’s instructions. The purity and concentration of total cellular RNA was determined using Nanodrop-1000, version 3.6.0. The minimum RNA amount obtained was considered as a base for calculations to reverse transcribe complementary DNA (cDNA) using the Verso cDNA kit (Thermo Scientific, USA) according to manufacturer’s protocols. The forward and reverse primers for the selected genes were designed from Integrated DNA Technologies (IDT, USA) and are listed in Table 1. Expression was quantified using real time RT-PCR analysis with SYBR green master mix kit (Applied Biosystems, USA). Data analysis was carried out using Applied Biosystems StepOne Plus thermal cycler and detection system. The real time RT-PCR analysis was carried out for two independent experiments with each sample run in duplicates. The gene expression levels were normalized to the expression of the housekeeping gene GAPDH and were expressed as fold changes relative to the expression of the genes in cells.