Nanodrop spectral photometer

The JPC-seeded scaffolds were placed in Lysing Matrix D microtubes with ceramic beads (MP Biomedicals, Irvine, CA, USA) and lysis buffer (Macherey-Nagel, Dueren, Germany), and shredded by a FastPrep-24 device (MP Biomedicals, Irvine, CA, USA). RNA isolation from the obtained lysates of JPC-seeded scaffolds was carried out using the NucleoSpin RNA Mini kit (Macherey-Nagel, Dueren, Germany) following the manufacturer’s instructions. After RNA quantification using the Nanodrop spectral photometer (Thermo Fisher Scientific, Waltham, USA), 200 ng of RNA was synthesized to cDNA using the SuperScript VILO kit (Thermo Fisher, Darmstadt, Germany) following the manufacturer’s instructions. mRNA transcription levels were quantified by a real-time LightCycler system (Roche Diagnostics, Mannheim, Germany). DNA Master SYBR Green 1 (Roche, Mannheim, Germany) and the primer kits (Search LC, Heidelberg, Germany) for the target genes (ALPL, COL1A1, RUNX2, OCN, GAPDH) were used for the PCR reactions. 35 cycles of amplification were carried out for each mRNA. The ratio of target gene copy numbers to those of the housekeeping gene (GAPDH) were calculated.

RNA isolation was performed as described previously [39] (link). In short, RNA from whole blood samples collected in PAXgene Blood Tubes (BD Biosciences, Franklin Lakes, NJ) was isolated using PAXgene Blood miRNA Kit (Qiagen, Hilden, Germany) according to manufacturers’ instructions in a semi-automated fashion using Qiacube robot (Qiagen) to ensure reproducibility. RNA concentration was measured with Nanodrop spectral photometer (ThermoFisher Scientific, Waltham, MA) and RNA Integrity was assessed using RNA Nano Bioanalyzer Kit (Agilent Technologies, Santa Clara, CA).