Danish University Colleges
Validation of an assay for telomere length measurement
Telling, Katrine Eidorf; Jørgensen, Malene Munk; Madsen, Helen Nordahl
Publication date:
2021
Link to publication
Citation for pulished version (APA):
Telling, K. E., Jørgensen, M. M., & Madsen, H. N. (2021). Validation of an assay for telomere length measurement. Poster session presented at The 34rd World Congress of the International Federation of Biomedical Laboratory Science, Copenhagen, Denmark.
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Download date: 05. Jul. 2022
Validation of an assay
for telomere length measurement
1 Epel ES, Blackburn EH, Lin J, Dhabhar FS, Adler NE, Morrow JD, Cawthon RM: Accelerated telomere shortening in response to life stress. National Academy of Sciences of the United States of America 2004, 101(49):17312-17315.
2 Cawthon RM: Telomere length measurement by a novel monochrome multiplex quantitative PCR method. Nucleic Acids Research 2009, 37(3): e21.
Telomere length (kbp)
0 30
10 40 60
20 50 70
0.5 µM telomere primers
Sample 1 Sample 2 Sample 3
1 µM telomere primers 5 µM telomere primers
Relative telomere length
Passage number
Figure 2: Relative telomere length in relation to cell culture passage number. Each result is based on five independent measurements of cell cultures with and without rapamycin treatment. Standard errors are added.
0 0,6
0,2 0,8 1,2
0,4 1
33 47
21
Figure 1: Measured telomere lengths when using different concentrations of telomere primers. Reference gene primers are used in the concentration of 0.5 µM.
Katrine Eidorff Telling1,2, Malene Munk Jørgensen1, Helen Nordahl Madsen1
1 School of Biomedical Laboratory Science, VIA University College, Aarhus N, Denmark 2Department of Pathology, Aarhus University Hospital
Introduction
Telomeres are repetitive sequences of the nucleotides TTAGGG which form and protect the ends of chromosomes. Telomeres
shorten with advanced age. In addition, adverse health
conditions and cellular stress have been shown to accelerate
this shortening.1) Therefore, the length of telomeres may be used as a measure for aging and stress. There are several potential
applications of telomere length (TL) measurements in research and diagnostics. The quantitative polymerase chain reaction
(qPCR) method is cheaper and faster than the less feasible gold standard method terminal restriction fragment (TRF) analysis for TL measurement, which makes TL-qPCR the preferable marker in large-scale clinical studies.The aim of this study was to validate and adjust our protocol for a TL-qPCR assay.
Materials and methods
The Stratagene Mx3000P Real-Time PCR System and SYBR
Green dye was used to determine the length of telomeres. Since the method is challenged by the repeated nucleotide sequence, which induces primer dimer formation, we used primers
specifically designed to avoid this.2) To validate the assay, the two following experiments were conducted. First, the effect of varying the telomere primer concentrations was examined in the range from 0.5 µM to 5.0 µM using two concentrations, 0.5 µM and 1.0 µM, of the reference gene primer. The
sample material was human fibroblast cells and human blood mononuclear cells. In the second experiment, six human
fibroblast samples from three different passages of cell cultures were used to compare passage number with telomere length.
Results
Figure 1 shows that the assay had a strong sensitivity to changes in primer concentration. The impact of varying telomere primer concentration had major impact on transcript level compared to changes in reference gene (36B4) primer concentrations. As seen in figure 2, telomere length declined with increasing cell culture passage number indicating telomere shortening among the most aging cells.
Conclusion
The experiment showed a robust assay demonstrating
reproducible results. As expected, shortening of telomere length was found in aging cells. TL-qPCR can be used for research
purposes if the primer concentrations are held cautiously constant due to the large impact on transcript level.
