Sybr one step rt pcr master mix

Total RNA was extracted from osteoblasts using a TRIzol kit (MDBio, Taipei, Taiwan). RNA quality and yield were determined by absorbance at 260-nm measurements performed with a Nanovue Spectrophotometer (GE Healthcare, Madison, WI). Complementary DNA was synthetized from 1 μg total RNA using a Moloney Murine Leukemia Virus Reverse Transcription kit (Invitrogen), following the manufacturer’s recommendations. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was carried out with a SYBR One-Step RT-PCR Master Mix (Applied Biosystems, Foster City, CA). All target gene primers were purchased from Applied Biosystems. qPCR assays were carried out in triplicate using a StepOnePlus sequence detection system (Applied Biosystems), according to the manufacturer’s instructions. All results are obtained from six independent experiments performed in duplicate.

Total RNA was extracted from osteoblasts using a TRIzol kit (MDBio, Taipei, Taiwan). RNA quality and yield were determined by absorbance at 260-nm measurements performed with a Nanovue Spectrophotometer (GE Healthcare, Madison, WI, USA). Complementary DNA was synthetized from 1 μg total RNA using a Moloney Murine Leukemia Virus Reverse Transcription kit (Invitrogen) following the manufacturer’s recommendations. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was carried out with SYBR One-Step RT-PCR Master Mix (Applied Biosystems, Foster City, CA, USA). All target gene primers were purchased from Applied Biosystems and the sequences were as follows: GAPDH, forward-AGCCACATCGCTCAGACAC, reverse-GCCCAATACGACCAAATCC; OSM, forward-AGTACCGCGTGCTCCTTG, reverse-CCCTGCAGTGCTCTCTCAGT. qPCR assays were carried out in triplicate using a StepOnePlus sequence detection system (Applied Biosystems), according to the manufacturer’s instructions. All results are expressed for six independent experiments performed in duplicate.