1st strand cdna synthesis system kit

Total RNA was isolated using the mirVana^™ miRNA isolation kit (Life technologies) according to the manufacturer’s instructions. For end-point PCR and real-time PCR, cDNA was generated using the 1^st strand cDNA synthesis system kit (OriGene) following the manufacturer’s suggestion. The PCR analysis was performed using the GeneAmp fast PCR master mix (Life technologies) as per manufacturer’s protocol. All primers were synthesized by Integrated DNA Technologies. The primer for mouse SIRT6 was forward (5-AGA GGA ATG TCC CAA GTG TAA G-3) and reverse (5-AAA GAC ACC AGG GTG AGA TAT G-3). Q-PCR analysis was performed using the OriGene qSTAR SYBR green kit following the manufacturer’s suggestion. The relative expression for osteocalcin (Ocn) mRNA was estimated from triplicate Q-PCR reactions following normalization to the β2-microglobulin. All primers for Q-PCR assay were purchased from OriGene.

M-CSF-dependent BMMs were prepared from the femurs and tibias of 4-to-6 week old wt and mutant male mice and cultured with 50 ng/ml M-CSF (Peprotech) and 100 ng/ml RANKL (Peprotech) for three days. Total RNA with small RNA (< 200 nucleotides) was isolated using the mirVana^™ miRNA isolation kit (Life technologies) according to the manufacturer’s instructions. For qRT-PCR, cDNA was generated using the 1^st strand cDNA synthesis system kit (OriGene) following the manufacturer’s suggestion. QRT-PCR was performed using the OriGene qSTAR SYBR Green Kit on an Mx4000 multiplex quantitative PCR System (Stratagene). The relative expression for c-Fos, NFATc1, and cathepsin K mRNA were estimated from triplicate qRT-PCR reactions following normalization to the β₂ microglobulin. All primers were purchased from the OriGene.