qPCR was performed as previously described.39 (link) Briefly, total RNA was isolated from samples using RNAeasy kit (Qiagen). DNAse I (Thermo-Fisher)-treated RNA was used to generate cDNA by reverse transcription according to the manufacturer’s instructions (First Strand cDNA Synthesis Kit; Roche Applied Science). qPCR reactions were performed in a BioRad CFX detection system using SYBR green PCR reagents as described by the manufacturer. A calibration curve was performed for each primer pair, and all oligo-nucleotides were tested to ensure specificity and sensitivity. Ribosomal RNA (18S) was used as an endogenous control. The expression levels of the target genes in each sample were calculated by normalizing the mRNA level of a particular gene against 18S ribosomal RNA, which is considered a stable housekeeping gene. The following oligonucleotide primer pairs were used:
18SForward: 5′ AGTCCCTGCCCTTTGTACACA
Reverse: 5′ CGATCCGAGGGCCTCACTA
Alkaline phosphatase(ALP)Forward: 5′ ACTGATGTGGAATACGAACTGGATGAGAAGG
Reverse: 5′ CAGTCAGGTTGTTCCGATTCAATTCATACTGC
Collagen type IForward: 5′ AACCTGGTGCGAAAGGTGAA
Reverse: 5′ AGGAGCACCAACGTTACCAA
Osteocalcin (OCN)Forward: 5′ TCTCTCTGACCTC ACAGATGCCAAGC
Reverse: 5′ GGACTGAGGCTCCAA GGTAGCG
β-ActinForward: 5′ TCACCCACACTGTG CCCATCTACGA
Reverse: 5′ CAGCGGAACCGCTC ATTGCCAATGG.