Abi quantstudio 6 flex rt pcr system

Total RNA was extracted from cells using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The same quantity of total RNA was reverse-transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA). Quantitative RT-PCR was performed using the ABI QuantStudio 6 Flex RT-PCR system with the TaqMan Universal PCR Master Mix (Applied Biosystems). The primers used in this study were c-Fos (Mm00487425_m1), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1, Mm00479445_m1), dendrocyte expressed seven transmembrane protein (DC-STAMP, Mm01168058_m1), ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d2 (Atp6v0d2, Mm00656638_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Mm99999915_g1) from the TaqMan Gene Expression Assay (Applied Biosystems). All reactions were run in triplicate. Relative expression of the target genes was calculated using the ΔΔCt method with GADPH gene as internal reaction control and expressed as fold change relative to the control untreated with RANKL and WELC. Experiments were repeated three times, and results from one representative experiment were shown.

RT-PCR was used to quantitatively describe the mRNA expression of TGF-β1, collagen I and collagen III. Total RNA was extracted with TRIzol Reagent (Ambion, Austin, TX, United States) according to the manufacturer’s instructions. RT-PCR was performed on an ABI QuantStudio 6 Flex RT-PCR System (Applied Biosystems, United States) with the SYBR Green I incorporation method. The relative expression levels of mRNAs were calculated using the 2^–ΔΔCt method. GAPDH was used as the internal control. The primers for related genes are listed in Table 1.