Real-time PCR analysis of SIRT1 mRNA expression
BLOOD
SIRT1 mRNA expression in the blood was evaluated by outsourcing the analysis to the Research Center for Immunological Analysis (Okayama, Japan). Briefly, whole blood was collected in a PAXgene RNA blood collection tube (Qiagen, Mississauga, Canada). Total RNA was isolated from the blood using a PAXgene Blood RNA Kit (Qiagen) and stored in RNase-free water at − 80 °C. First-strand cDNA was reverse-transcribed with total RNA using a T100 Thermal Cycler (Bio-Rad, CA, USA). The mRNA expression level of the target genes was measured using a CFX384 Touch Real-Time PCR Detection System (Bio-Rad). 