Sybr light cycler 480 sybr green 1 master

alizarin red S staining was performed to confirm mineral deposition. Samples fixed with 4% paraformaldehyde (PFA) solution were stained with an alizarin red S (Sigma) solution of 2% w/v diluted by 2DW at pH 4.3 for 20 min. To quantify the mineral deposition, the stained samples were de-stained with 10% cetylpyridinium chloride solution in 10 mM sodium phosphate buffer (pH 7.0) overnight. The optical density of each de-stained sample was measured utilizing a microplate reader at 550 nm.
The messenger RNA (mRNA) expression of dentin matrix acid phosphoprotein 1 (DMP-1) and dentin sialophosphoprotein (DSPP) in each sample was quantitatively evaluated. For RNA extraction, each sample was treated with Trisure (Bioline, London, UK) for 10 min. The extracted RNA was used for complementary DNA (cDNA) synthesis using HelixCript™ Thermo Reverse Transcriptase (NanoHelix, Daejeon, South Korea). Biometra TProfessional TRIO Thermo-cycler (Analytik Jena AG, Jena, Germany) was used for the synthesis. The cDNA mixed with appropriate primers (Supplemental Appendix Table 1) was amplified with Roche SYBR Light Cycler 480 SYBR Green I Master (Roche Diagnostics Gmbh, Mannhein, Germany) using a light cycler 480 II (Roche Diagnostics GmbH). Expression levels of DMP-1 and DSPP were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) via the ΔΔCt method.