Nzyspeedy qpcr green master mix 2

Quantification of OB, CPT1a, MPC1, SLC1A5, GLS1, SLC2A4, LDHA, FABP4 and housekeeping genes β-actin and RPII expression was achieved by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, following the instructions given by NZYSpeedy qPCR Green Master Mix (2×) (MB22402, NZYTech, Lisbon, Portugal). No cDNA was added in the negative control. A duplicate was performed for each sample. After mix preparation, samples were placed in the CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA, USA). The three-step cycling qRT-PCR program was performed as described in Table S4. The quantitative analysis was followed and obtained with the software CFX-Maestro™ (Bio-Rad, Hercules, CA, USA). The qRT-PCR data analysis was conducted using the comparative CT method. Individual relative gene expression values (fold change) were calculated using the following formula: 2^−ΔCt.

After 21 days of osteogenic differentiation, PDLSC, MSC(M) and MSC(AT) total RNA was extracted using RNeasy Mini Kit (QIAGEN, Hilden, Germany), followed by cDNA synthesis with High-Capacity cDNA Reverse Transcription kit (Life Technologies, Carlsbad, CA, USA). Primer sequences used are summarized in Table 1, and qRT-PCR was performed using NZYSpeedy qPCR Green Master Mix (2×), ROX plus (NZYTech, Lisbon, Portugal) and StepOnePlus real-time PCR system (Applied Biosystems, Waltham, MA, USA). Reactions were performed in triplicate and carried out for 10 min at 95 °C, followed by 40 cycles of 15 sec at 95 °C and 1 min at 60 °C. Gene expression was normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and fold-change was calculated considering baseline expression at day 0 (undifferentiated cells). A threshold cycle (Ct) was observed in the exponential phase. ΔΔCt values were calculated using geometric means and expressed as 2^−ΔΔCt.

Initially, cDNA samples were diluted 80× in sterile distilled water (B. Braun, Melsungen, Germany). Then, on ice, per each well of a 384-well plate, the following were added: 5 μl of NZYSpeedy qPCR Green Master Mix (2×) (NZYTECH, Portugal), 1 μl of miRNA specific primer mix (microRNA LNA™ PCR primer set, Exiqon), and 4 μl of previously diluted cDNA. Each amplification reaction was performed in triplicate on a LightCycler 480 instrument (Roche Diagnostics, Manheim, Germany). Each plate also contained two negative template controls. RT-qPCR protocol consisted of a denaturation step at 95ºC for 2 min, followed by 40 amplification cycles at 95ºC for 5 s and 60ºC for 20 s. Melting curve analysis was performed according to the instrument’s manufacturer’s recommendations.
SNORD38B was used as a reference gene for data normalization, as this gene was the most stably expressed over the whole range of the samples used for the global expression assay. Notwithstanding, the stability of SNORD38B expression was empirically validated in additional samples. Relative miRNA expression in each sample was calculated by the 2^−ΔΔCT method (the target sequences of mature miRNAs analyzed are provided in Supplementary Table 1).