Oct 01, 2024
Relative Telomere Length Measurement
- 1Vetmeduni Vienna
Protocol Citation: Sinan Gültekin 2024. Relative Telomere Length Measurement. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1rn7lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 01, 2024
Last Modified: October 01, 2024
Protocol Integer ID: 108714
Abstract
Relative Telomere Length Measurement by qPCR
Relative Telomere Length Measurement
Relative Telomere Length Measurement
DNA isolation
Genomic
DNA was extracted using the DNeasy Blood and Tissue Kit
(Qiagen, Hilden, Germany
qPCR
qPCR
10 ng/μL DNA was amplified in a 5x EvaGreen‱ mix (No ROX) (Bio&Sell, Nurnberg, Germany). The thermal cycling protocol of Telomere A: 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′, Telomere B: 5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′ and B2M was: 95°C for 15 min, followed by 40 cycles of 95°C for 15 s, 56°C for 20 s, and 72°C for20 s. The thermal cycling protocol of Telomere Primer Pair 1 was:95°C for 15 min, followed by 2 cycles of 15 s at 94°C, 1 cycle of 15 s at 49°C,40 cycles of 15 s at 94°C, 1 cycle 10 s at 62°C, 1 cycle 15 s at 74°C with signal acquisition, 10 s at 84°C, and 15 s at 88°C with signal acquisition. A no-template control (NTC) and the all templates were run in triplicate for each
reaction
Primer sequences
Relative telomere length (RTL) was determined using two different primer pairs for telomere amplification and a primer pair for the single-copy reference gene beta-2-microglobulin(B2M). The following primers were employed: Telomere Primer Pair 1 (Hermann C, 2023): Telg: 5′-ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGTTAGTGT-3′,Telc:
5′-TGTTAGGTATCCCTATCCCTATCCCTATCCCTATCCCTAACA-3′. Telomere Primer Pair 2 (Mugdha, 2020): Telomere A: 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′, Telomere B: 5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′. Single Copy Gene: Beta-2-microglobulin (B2M): Forward (F): 5′-CAAGACACCCGCCAGAAGAT-3′, Reverse (R): 5′-CAGCGTGGGACAGAAGGTAG-3′ (Designed by our lab).
Data analysis
Data analysis
Data were analyzed using ViiA7 software (ThermoFisher, MA, USA), and RTL was calculated using the ΔΔCt method: ΔΔCt = (Sample B2M Ct - Sample Telomere Ct) - (Control B2M Ct - Control Telomere Ct).