DNA isolation was performed by using DNA Purification System kit (Promega, Fitchburg, WI, USA) according to the manufacturer’s protocol. Total of 20 mg of tissue were digested overnight and subsequently, samples were transferred to minicolumns and centrifuged. Then, minicolumns were washed and DNA samples were eluted.
The average telomere length ratio (ATLR) was measured from total genomic DNA using qRT-PCR method as reported by Callicott et al. [19 (link)]. Telomeres primers and 36B4 gene (as internal control) were used and 36B4 gene was employed as a reference for standard curve construction (from 3.75 to 90 ng). Forward telomeres primer sequence used for telomere length was: 5′ CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT 3′. Reverse primer sequence was: 5′ GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT 3′. Forward and reverse primers sequences for 36B4 gene were respectively: 5′ ACT GGT CTA GGA CCC GAG AAG 3′ and 5′ TCA ATG GTG CCT CTG GAG ATT 3′. For telomere and 36B4 assays, 100 nM of both forward and reverse primers, 12.5 μL Syber Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) were used. Reaction conditions were initial denaturation step at 95 °C for 10 min, followed by 30 cycles of 95 °C for 15 s, and a 56 °C anneal-extend step for 1 min for data collection.
The average telomere length ratio (ATLR) was measured from total genomic DNA using qRT-PCR method as reported by Callicott et al. [19 (link)]. Telomeres primers and 36B4 gene (as internal control) were used and 36B4 gene was employed as a reference for standard curve construction (from 3.75 to 90 ng). Forward telomeres primer sequence used for telomere length was: 5′ CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT 3′. Reverse primer sequence was: 5′ GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT 3′. Forward and reverse primers sequences for 36B4 gene were respectively: 5′ ACT GGT CTA GGA CCC GAG AAG 3′ and 5′ TCA ATG GTG CCT CTG GAG ATT 3′. For telomere and 36B4 assays, 100 nM of both forward and reverse primers, 12.5 μL Syber Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) were used. Reaction conditions were initial denaturation step at 95 °C for 10 min, followed by 30 cycles of 95 °C for 15 s, and a 56 °C anneal-extend step for 1 min for data collection.