Standard real time pcr thermal cycler

Total RNA was isolated from rat brain using the innuPREP RNA Mini Kit (Analytik Jena AG, Germany). The mRNA concentration was determined on a DS-11 Spectrophotometer/Fluorometer (DeNovix, USA). The Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., USA) was used to synthesize cDNA samples for subsequent RT-PCR on a Standard real-time PCR Thermal Cycler (Analytik Jena AG, Germany). Specific primer sequences for Vdr, Vdbp, Cyp27b1, Cyp24a1, Nf-κb, Iκb-alpha, and the glyceraldehyde 3-phosphate dehydrogenase (Gapdh) reference gene were designed using Primer BLAST software and used at a working concentration of 10 μM:
Target genes were amplified for 60 cycles using Maxima SYBER Green/ROX qPCR Master Mix (Thermo Fisher Scientific Inc., USA). Relative mRNA expression calculations were performed according to the 2^–ΔΔCt comparison method. The expression level of each gene was normalized for GAPDH in the same samples and then calculated as a fold change compared to the control.

Extraction of total RNA from bone tissue was performed using the innuPREP RNA Mini Kit (Analytik Jena AG, Germany). Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., USA) was used to synthesize cDNAs samples, which were served as templates for real-time PCR analysis on Standard real-time PCR Thermal Cycler (AnalytikJena AG, Germany). Specific primer sequences for Vdr, Cyp27b1, and a reference gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh) were designed using Primer BLAST software: Vdr–forward 5′-TCATCCCTACTGTGTCCCGT-3´; reverse 5′-TGAGTGCTCCTTGGTTCGTG-3´; Cyp27b1–forward 5′-TGGGTGCTGGGAACTAACCC-3; reverse 5′-TCGCAGACTGATTCCACCTC-3´; Gapdh–forward 5′-TGAACGGGAAGCTCACTGG-3´; reverse 5′-TCCACCACCCTGTTGCTGTA-3´. Target genes were amplified for 60 cycles using Maxima SYBER Green/ROX qPCR Master Mix (Thermo Fisher Scientific Inc., USA). Calculations of relative mRNA expression were performed according to the comparative 2−ΔΔCt method of Livak and Schmittgen [14 (link)]. Data were normalized to an internal housekeeping gene Gapdh and then calculated as the fold change relative to control.