Taqman random hexamers

Femur and tibia bone marrow were flushed with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and calvaria, livers, tibias, and ileums were flash frozen, pulverized, and homogenized in TRIzol reagent. Cultures were washed twice with 1X PBS, and TRIzol reagent was directly applied. RNA was isolated by the TRIzol method following the manufacturer's instructions. Total RNA was quantified via NanoDrop 1000 (Thermo Fisher Scientific). cDNA was synthesized using TaqMan random hexamers and reverse transcription reagents (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. cDNA was amplified using TaqMan gene expression primers/probes and universal PCR master mix via the StepOnePlus System (Applied Biosystems). Gapdh was used as an endogenous control for studies in marrow, calvaria, tibias, and ileums. Gapdh and Rn18s were used as internal controls for studies in livers. Relative quantification of data was performed via the comparative C_T method (2^−ΔΔCT)49 as previously described.9, 13

cDNA was synthesized utilizing TaqMan Random Hexamers and Reverse Transcription Reagents (Applied Biosystems, Waltham, MA, USA), per the manufacturer’s protocols, and was amplified using TaqMan primer probes and TaqMan Fast Advanced Master Mix on the StepOnePlus System (Applied Biosystems), per the manufacturer’s protocols. GAPDH was used as an endogenous control gene, and relative quantification of mRNA was performed by the 2^−ΔΔCT method. Assay was performed with two technical replicate reactions.