Quantitative PCR Analysis of Osteogenic Markers

The expression of select osteogenic marker genes was determined by quantitative PCR (qPCR) essentially as previously described, with some minor modifications^{34 (link)}. In brief, total RNA was extracted from 7F2 cells using a modified version of a hybrid Tri Reagent (Sigma-Aldrich, USA)/RNEasy® protocol. RNA was quantified using a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA), and concentrations were brought to a uniform level using additional RNase-free water. RNA was reverse transcribed to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The cDNA was amplified in TaqMan Fast universal PCR master mix with TaqMan assay primers and probes according to the manufacturer’s instruction. Genes of interest were: ALPL (Mm00475834_m1), RUNx2 (Mm00501584_m1) and Sparc/osteonectin/BM40 (Mm00486332_m1). Quantitative PCR (qPCR) was performed in a RealPlex Real-Time PCR System (Eppendorf, Enfield, CT) with fast thermal cycling as described by Taqman (Applied Biosystems). The level of expression of each gene was normalized to the level of expression of a common standard housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to determine the fold change in up/downregulation of the genes of interest using the comparative CT method (2-ΔΔCT)^{35 (link)}.