Total RNA was isolated using TRIzol reagent protocol (Invitrogen) and a treatment with DNase I amplification Grade (Sigma-Aldrich, St. Louis, MO, USA) was done according to the manufacturer’s instruction. Quantification was performed by spectrophotometry (ND-1000, Nanodrop Technologies, Wilmington, DE, USA). The sequence of primers used for RT-PCR is shown in Table 1 . Quantification was done using the QuantiTect SYBR Green RT-PCR kit (Qiagen GmbH, Hilden, Germany) for 50 µg of RNA and 1 µl of primer. The mRNA expression was analyzed in the Light Cycler thermal cycler system (Roche Diagnostics, Indianapolis, IN, USA) and the relative expression of the target genes was determined using the 2−ΔΔCt method.
Sequences of the primers used for real-time RT-PCR.
| Gene | Forward primer (5′-3′) | Reverse primer (5′-3′) |
|---|---|---|
| FGF23 | TGGCCATGTAGACGGAACAC | GGCCCCTATTATCACTACGGAG |
| RunX2 | CGGGAATGATGAGAACTACTC | GCGGTCAGAGAACAAACTAGGT |
| Osterix | GTACGGCAAGGCTTCGCATCTGA | TCAAGTGGTCGCTTCGGGTAAAG |
| STIM1 | CAGTACTACAACATCAAGAAGC | TTTTTATTTTCTCAGCCCCC |
| Tbp | ACTCCTGCCACACCAGCC | GGTCAAGTTTACAGCCAAGATTCA |
All primers were purchased from Eurofins Genomics Germany GmbH, Ebersberg, Germany except STIM 1 which was purchased from Sigma-Aldrich, St. Louis, MO, USA.
FGF23, fibroblast growth factor 23; RunX2, Runt-related transcription factor 2; STIM1, Stromal interaction molecule 1; Tbp, TATA sequence binding protein.