Thermal cycle dice real time system tp800

Total RNA was extracted from brain and skin tissues using an ISOGEN kit (NIPPON GENE, Tokyo, Japan). RNA (1 μg) samples were reverse-transcribed into cDNA using the PrimerScript RT reagent Kit with gDNA Eraser (Takara Bio, Otsu, Japan). cDNA samples from each tissue were amplified using the KAPA SYBR FAST Universal qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) on a Thermal Cycle Dice Real Time system TP800 (Takara Bio, Otsu, Japan). PCR primers used for the detection of fLuc and NG2 were as follows: forward (5′-ACAACCGCGAAAAAGTTGCG-3′), reverse (5′-TGCGTCGAGTTTTCCGGTAAG-3′) for fLuc and forward (5′-ACAACACCACACCCAAGACAC-3′), reverse (5′-TCAAGCTGCATCCATACTG-3′) for NG2.

RAW264.7 cells were seeded at 1.0 × 10⁵ /well in six-well plates with 50 ng/mL RANKL for 3 or 5 days. Total RNA was extracted using the ReliaPrep™ RNA Cell Miniprep System (Promega, Madison, WI, USA). Total RNA (0.5 μg) was reverse-transcribed using the SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen ThermoFisher Scientific) according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa Bio, Shiga, Japan) and gene-specific primers with the Thermal Cycle Dice Real-Time System TP800 (TaKaRa Bio). Reactions were performed in duplicate, and relative mRNA levels were calculated by the comparative threshold cycle method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. The primer sequences were as follows: GAPDH (forward, 5′-TGTGTGTCCGTCGTGGATCTGA-3′; reverse, 5′-TTGCTGTTGAAGTCGCAGGAG-3′^{35 (link)}); TRPV2 (forward, 5′-AGGAGCTGACTGGACTGCTA-3′; reverse, 5′-GAGCCTTCTGTGTATGCCGA-3′^{28 (link)}); TRPV4 (forward, 5′- CCACCCCAGTGACAACAAG-3′; reverse, 5′- GGAGCTTTGGGGCTCTGT-3′^{28 (link)}); NFATc1 (forward, 5′-CCCGTCACATTCTGGTCCAT-3′; reverse, 5′-CAAGTAACCGTGTAGCTGCACAA-3′^{36 (link)}); Cathepsin K (forward, 5′-AGGCAGCTAAATGCAGAGGGTACA-3′; reverse, 5′-AGCTTGCATCGATGGGACACAGAGA-3′^{37 (link)}); and TRAP (forward, 5′-AGCTTGCATCGATGGGACACAGAGA-3′; reverse, 5′-GTCAGGAGTGGGAGCCATATG-3′^{37 (link)}).

Total RNA was isolated from atria using TRI reagent (Molecular Research Center). Reverse transcription was performed using ImpromII reverse‐transcriptase (Promega) with oligo‐dT priming. PCR was performed using a Thermal Cycle Dice Real Time System TP800 (Takara) with SYBR Green (Takara) as a fluorescent dye. The primers used in this study are shown in Table S1.