Tp800 pcr thermal cycler dice detection system

Total RNA were isolated as described previously using TRIzol reagent and reverse transcribed34 (link)37 (link). RNA integrity was assessed by light absorbance at 260 and 280 nm and by agarose gel electrophoresis with ethidium bromide staining. Real-time RT-PCR was performed using an SYBR Premix Ex Taq kit (TaKaRa Biotechnology, Otsu, Japan) and a TAKARA TP800 PCR Thermal Cycler Dice Detection system at 95 °C for 10 min for initial denaturation, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. All reactions were performed in triplicate and analyzed by the 2-ΔΔCT method38 (link). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control. The gene-specific primers for osteocalcin (OCN), Runx2, Osterix, β-catenin, axin-2, and GAPDH were as follows: OCN forward 5′-TCCCACACAGCAGCTTGGCCC-3′ and reverse 5′-TGAGGCTCCAAGGTAGCGCCG-3′; Runx2 forward 5′-TTGACCTTTGTCCCAATGC-3′ and reverse 5′-AGGTTGGAGGCACACATAGG-3′; Osterix forward 5′-TGAGCTGGAACGTCACGTGC-3′ and reverse 5′-AAGAGGAGGCCAGCCAGACA-3′; β-catenin forward 5′-ACGGTGCCGCGCCGCTTATA-3′ and reverse 5′-TAGCCATTGTCCACGCAGCGG-3′; axin-2 forward 5′-GTCTCTACCTCATTTCCCGAGAAC-3′ and reverse 5′-CGAGATCAGCTCAGCTGCAA-3′; GAPDH forward 5′-GAGAAGGCTGGGGCTCATTT-3′ and reverse 5′-CCAATATGATTCCACCCATG-3′.

The distal femur tissues were collected from six rats in each group and cryopreserved in liquid nitrogen. After weighing, the distal femurs were ground into powder in a mortar with added liquid nitrogen. Then, we added 8 mL TRIzol (Sigma) after the liquid nitrogen volatilizes to extract total RNA. A miRNA Isolation kit (Ambion) was used to isolate total miRNA according to the manufacturer recommendation. Quantitative RT‐PCR analysis for miRNAs was performed using a TP800 PCR Thermal Cycler Dice Detection System (Takara) and SYBR RT‐PCR kits (Takara). Stem‐loop primers for miR‐106b and U6 were brought from Ambion.