Abi prism 7300 thermocycler

Cells were suspended in 700 μl TRIzol reagent (ThermoFischer Scientific) and RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The concentration of total RNA was measured using Nanodrop. Using the high capacity cDNA reverse transcription kit (Applied Biosystems), total RNA was converted to cDNA. Levels of mRNA were assessed by quantitative real-time PCR (qPCR) analysis with a Fast SYBR green master mix (Applied Biosystems) on an ABI Prism 7300 thermocycler (Applied Biosystems). Expression of human Rα1, Rα2, TSS1- and TSS2-specific Rα2, and periostin and murine Rα1 and Rα2 was assessed using sequence-specific primers listed in Supplementary Table 2. Unless specified otherwise, the human Rα2 primer employed was designed to bind downstream of exon 3 and is common to both TSS1- and TSS2-initiated transcripts. Relative gene expression was determined by the ΔCT method, and GAPDH and β-actin were used as a reference gene for human and mouse samples, respectively.

Total RNA was extracted with TRIzol (Life Technologies). Reverse transcription was performed using a High-Capacity cDNA Archive Kit (Life Technologies). Quantitative real-time PCR was performed using Power SYBR^® Green Master Mix (Thermo Scientific), in an ABI PRISM 7300 thermocycler (Applied Biosystems). Variations in RNA input were corrected using the expression of the GAPDH housekeeping gene. The ΔΔCT method was used for relative quantification.

Total RNA was extracted with Trizol (Life Technologies). Reverse transcription was performed using random priming and Superscript Reverse Transcriptase (Life Technologies). Quantitative real-time PCR was performed using Absolute SYBR Green mix (Thermo Scientific) in an ABI PRISM 7300 thermocycler (Applied Biosystems). Variations in input RNA were corrected by subtracting the number of PCR cycles obtained for GAPDH.

Real-time RT-PCR was performed for HSP70, Runx2, Osterix, and GAPDH using SYBR Green RT-PCR Master Mix (Life Technologies). Real-time quantitative RT-PCR was performed using the ABI Prism 7300 Thermocycler (Applied Biosystems) following the manufacturer’s instructions. The thermal profile for real-time PCR was 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative gene expression levels from real-time PCR experiments were assessed using the 2^−∆∆CT method^{47 (link)}. Primers for human Runx2, Osterix, and GAPDH are AAATCGCCAGGCTTCATA (forward)/CTGCCAGGAGTGGTCAAA (reverse); CCTGCGACTGCCCTAATT (forward)/GCGAAGCCTTGCCATACA (reverse); and GGATTTGGTCGTATTGGG (forward)/GGAAGATGGTGATGGGATT (reverse) respectively^{2 (link)}.