DNA sample collection, extraction, amplification, and measurement

About 3 ml of venous blood sample was collected from the subjects participating in the study in an EDTA bulb,[13,14] and processed immediately for DNA isolation and for assessing the TL by Real-time polymerase chain reaction (PCR) as per the standard procedure. The Qiagen DNA Mini Kit (QIAGEN Inc., California, United States Lot no. 163032024 manufactured in 2019) was used to extract the DNA from the peripheral blood leukocytes.[15] After the DNA was isolated its TL was measured using quantitative RT-PCR as T/S ratio. Telomere standard curve was generated by plotting CT (cycle threshold) values against the amount of telomere sequence in kb per reaction [Figure 1]. Cycling conditions for both the Telomere and 36B4 amplicons were as follows: 10 min at 95°C followed by 40 cycles at 95°C for 15s and 60°C for 1 min, followed by a melt curve. The 36B4 is a Single-Copy Gene and serves as a reference gene in the conventional qPCR technique in the measurement of Telomeres [Figure 2].[14] All samples were analysed in the ABI Step One Plus RT-PCR System with SDS version Step One Plus software.[13]

Standard curve for polymerase chain reaction cycles (generated by plotting cycle threshold values against the amount of telomere sequence in kb per reaction)

Single copy gene standard curve