Icycler iq5 optical system

Total RNA was extracted from the muscle biopsies and integrity assessed as described above. For reverse transcription-PCR (RT-PCR) analysis, 0.5 μg of total RNA was used in the reaction with SuperScript reverse transcriptase (Life Technologies). The resulting cDNA was analyzed by realtime quantitative PCR (RTqPCR) performed with the iQ5- iCycler Optical System (Bio-Rad, Hercules, CA, United States). IQ SYBR Bio-Rad protocol (100 mM KCl, 40 mM Tris–HCl pH 8.4, 0.4 mM dNTPs, iTaq DNA polymerase 50 U/ml, 6 mM MgCl2, SYBR Green I, 20 nM fluorescein, stabilizer) was applied according to the manufacturer’s instructions. Reaction mixtures were incubated at 95°C for 30 s, followed by two cycles at 95°C for 30 s and 95°C for 3 min and by 40 cycles at 95°C for 15 s and 60°C for 1 min. Finally, 80 cycles were run starting at 55°C and increasing the temperature by 5°C every 10 s up to 95°C. Fluorescence signals were measured during the elongation step. All measurements were performed in duplicate. The target mRNA expression levels were normalized to the levels of the polymerase (RNA) II (DNA directed) polypeptide A (Pol2A) gene using the 2^-ΔΔCT method as described in (Vitucci et al., 2018 (link)). The Oligonucleotide primer sequences used in RTqPCR are reported below:
      

Freshly isolated MPCs, P2-MSCs, and HUVECs were processed for gene expression analysis of endothelial-associated genes (PECAM, vWF, CDH5, KDR, TEK, TIE1, DLL4, and JAG1), mesenchymal/pericyte-associated genes (ACTA2, DES, CSPG4, RUNX2, TEM1, MCAM, RGS5, and LEPR), MPC-related genes (NES, OCT-4A, SPP1, NANOG, GP130, LIFR, and RANK), and cytokines (SCF, ANGPT1, ANGPT2, PDGFA, PDGFB, and RANKL). Total RNA was extracted using the RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. RNA samples (100 ng) were retro-transcribed using a QuantiTect® Reverse Transcription Kit (Qiagen) and 2 μl samples of 10-fold cDNA dilutions were amplified by quantitative real-time PCR (qRT-PCR), using the iCycler-iQ5 Optical System (Bio-Rad, Hercules CA, USA) and SsoAdvancedSYBR Green SuperMix (Bio-Rad). Samples were run in duplicate. All primer pairs (see Additional file 1) were from Sigma-Aldrich. Relative quantitative analysis was performed following the 2^–ΔΔCt Livak method [12 (link)]. Normalization was performed using RPL13A and ACTB housekeeping genes. Hierarchical clustering analysis was performed by applying HeatmapGenerator 5 software [13 (link)]. Values were reported as mean of normalized fold expression ± SEM. Statistical analysis was carried out by two-tailed t test applying the Mann–Whitney test.