Genequant 1300 device

On day 5 of culture, total RNA was extracted using TRIzol LS reagent (Invitrogen). The extracted total RNA was purified using the SV Total RNA Isolation System kit (Promega, Madison, WI, USA). Then, it was quantified at different wavelengths (230, 260, 280, and 320 nm) in the GeneQuant 1300 device (GE Healthcare, Cardiff, WLS, UK) and assessed for its integrity using gel electrophoresis for the intact 28S and 18S ribosomal RNA (Figures S1 and S2). cDNA was synthesized from 1 µg of total RNA by reverse transcription using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). For real-time PCR, TaqMan probes (Applied Biosystems) (Table 1) were used in a CFX96 device (Bio-Rad, Hercules, CA, USA), with a final volume of 10 µL per reaction and 11.25 ng of cDNA. The results were analyzed based on the value of the cycle threshold (Ct), and the normalization and relative quantifications of gene expression were performed by the 2^−ΔΔCT method [25 (link)]. The data obtained were represented as the fold difference in the expression of the gene normalized to the constitutive gene (Gapdh), assigning a value of 1 to each marker in MC3T3-E1 cultures on the Control-Ti surface.

Briefly, the total RNA was removed using TRIzol LS (Invitrogen) and purified using the SV Total RNA Isolation System kit (Promega, Madison, WI, USA). Then, it was quantified in the GeneQuant 1300 device (GE Healthcare, Cardiff, UK) and assessed for its integrity using the 2100 Bioanalyser (Agilent, Santa Clara, CA, USA) (Figures S3 and S4). cDNA was synthesized from 1 µg of total RNA by reverse transcription using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s instructions. TaqMan probes (Applied Biosystems) (Table 2) were used to access the osteoblastic gene expression via the CFX96 device (Bio-Rad, Hercules, CA, USA), as previously detailed [12 (link)]. PCR reactions were performed in one biological replicate, resulting from the pooling of 16 independent wells/experimental replicates for each group, and with three technical replicates, at two different moments. The results were analyzed based on the value of the cycle threshold (Ct), and the normalization and relative quantification of the gene expression were performed by the 2^−ΔΔCT method [25 (link)]. The results were normalized by constitutive gene Gapdh, assigning 1 to the MC3T3-E1 group. No statistical testing was applied due to pooling [12 (link)].