Superscript 2

Total RNA was isolated using TRIzol® reagent reversely transcribed into cDNA using Superscript II reverse transcriptase (Toyobo Life Science, Osaka, Japan). The qRT-PCR reaction system contained 2 μL of cDNA sample solution, 10 μL of SYBR-Green PCR master mix, 0.5 μL of forward and reverse primers (1 μM), and 7.5 μL of H₂O. The primer sequences were as follows: SIRT1, forward 5′-GCC AGA GTC CAA GTT TAG AAG A-3′, reverse 5′-CCA TCA GTC CCA AAT CCA G-3′; FOXO1, forward 5′-GGC TGA GGG TTA GTG AGC AG-3′ and reverse 5′-AAA GGG AGT TGG TGA AAG ACA-3′ and GAPDH, forward 5′-CCT CAA GAT CAT CAG CAA TG-3′ and reverse 5′-CCA TCC ACA GTC TTC TGG GT-3′. The amplification process was carried out as follows: denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 45 s, annealing at 50 °C for 45 s and elongation at 72 °C for 45 s, with the final extension step maintaining at 72 °C for 10 min. qRT-PCR was performed using an ABI Prism 7500 Fast Real-time PCR instrument (Applied Biosystems; Foster City, CA, USA), and analyzed using the 2-ΔΔCt method. The mRNA levels of SIRT1 and FOXO1 were normalized to those of GAPDH to assess the significance of the differences between the groups.

Total RNA was extracted from the second cohort using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. cDNA was synthesized by random primers and Superscript II reverse transcriptase (Toyobo, Osaka, Japan). The primers used for amplification for THBS2: forward primer 5’-CGTGGACAATGACCTTGTTG-3’ and reverse primer 5’-GCCATCGTTGTCATCATCAG-3’. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified in the same q-PCR as an internal control using primers: forward primer 5’-AGCCACATCGCTCAGACAC-3’ and reverse primer 5’-GCCCAATACGACCAAATCC-3’. The reaction ran on the ABI 7900HT Sequence Detection System (Applied Biosystems, CA, USA) in the presence of SYBR-Green dye (Toyobo, Osaka, Japan). The reaction condition was a denaturation program (95°C for 5 min), and an amplification and quantification program for 40 cycles (95°C for 15 s and 60°C for 45 s). Every sample was tested in triplicates, and a melting curve analysis of each sample was used to check the specificity of amplification. The expression level was determined as a ratio between THBS2 and the internal control GAPDH in the amounts of mRNA calculated by comparative CT method.