Genequant

Cell differentiation was quantified in terms of odontogenic gene expression by collecting total RNA using TRIzol reagent at prescribed times. Isolated RNA was pelleted, washed in 75% ethanol, and resuspended in nuclease-free water. RNA concentration of each sample was measured spectroscopically by GeneQuant (GE Healthcare Life Sciences, Little Chalfont, UK), and one microgram of isolated RNA was then reverse-transcribed into complementary DNA (cDNA) using M-MLV reverse transcriptase in a 20 μL reaction system according to manufacturer's instruction. The resulting complementary DNA (cDNA) was used for real time RT-PCR. Real time RT-PCR was carried out using a LightCycler Nano (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instruction. The comparative 2^−ΔΔCt method was employed to calculate relative gene expression. The gene expression levels were normalized to the β-actin mRNA level. Primer sequences and reaction condition are described in Tables 1 and 2, respectively.

Total DNA was extracted from pelleted blood cells using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA concentrations and the 260 nm/280 nm ratio were measured spectrophotometrically using GeneQuant (GE Healthcare, Warsaw, Poland) and stored at −20 °C until use. The DNA quality of selected samples was tested using capillary electrophoresis with highly sensitive gel (Fragment Analyser, Agilent).

RNA was prepared using RNeasy Lipid Tissue Mini Kit (Qiagen, Izasa, Barcelona, Spain). The integrity of each RNA sample was checked by the Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Total RNA was quantified by a spectrophotometer (GeneQuant; GE Health Care, Piscataway, NJ, USA) reverse transcribed to cDNA with the High Capacity cDNA Archive Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Gene expression was assessed by real-time PCR with a LightCycler 480 Real-Time PCR System (Roche Diagnostics, Barcelona, Spain), using TaqMan and SYBR green technology suitable for relative gene expression quantification. TaqMan probes for Fasn, Pparg, Irs1, Adipoq, Lep, Plin1, Dgat1, Tfna, Il6, Cyba, Tfam, Nrf1, and 18S are detailed in Supplementary Material Table S2. The RT-PCR reaction was performed in a final volume of 12 μL. The cycle program consisted of an initial denaturing of 10 min at 95 °C then 40 cycles of 15 s denaturing phase at 95 °C and 1 min annealing and extension phase at 60 °C. Fold changes compared with the endogenous control were then determined by calculating 2^−∆Ct. The gene expression results are expressed as the expression ratio relative to 18S gene expression according to the manufacturer’s guidelines. No difference in the expression of 18S was observed between groups (p = 0.1848).