Effect of pre-analytic variables on the reproducibility of qPCR relative telomere length measurement

<div><p>Telomeres, long nucleotide repeats and a protein complex at chromosome ends, shorten with each cell division and are susceptible to oxidative damage. Quantitative PCR (qPCR) is a widely-used technique to measure relative telomere length (RTL) in DNA samples but is challenging to optimize and significant lab-to-lab variability has been reported. In this study, we evaluated factors that may contribute to qPCR RTL measurement variability including DNA extraction methods, methods used for removing potential residual PCR inhibitors, sample storage conditions, and sample location in the PCR plate. Our results show that the DNA extraction and purification techniques, as well as sample storage conditions introduce significant variability in qPCR RTL results. We did not find significant differences in results based on sample location in the PCR plate or qPCR instrument used. These data suggest that lack of reproducibility in published association studies of RTL could be, in part, due to methodological inconsistencies. This study illustrates the importance of uniform sample handling, from DNA extraction through data generation and analysis, in using qPCR to determine RTL.</p></div

Correlation of relative telomere length (standardized T/S ratio) of matched subjects across extraction techniques and assay techniques.

<p>Inset heat map displays coefficient of determination (R²) for each correlation.</p

Correlation of relative telomere length (standardized T/S ratio) of matched samples pre- and post-purification.

<p>(Top) All samples by purification technique. (Bottom) By purification technique and extraction technique, shown by color, for 10 matched subjects extracted using three different techniques.</p

Correlation of Leukocyte Telomere Length Measurement Methods in Patients with Dyskeratosis Congenita and in Their Unaffected Relatives

Several methods have been employed to measure telomere length (TL) in human studies. It has been difficult to directly compare the results from these studies because of differences in the laboratory techniques and output parameters. We compared TL measurements (TLMs) by the three most commonly used methods, quantitative polymerase chain reaction (qPCR), flow cytometry with fluorescence in situ hybridization (flow FISH) and Southern blot, in a cohort of patients with the telomere biology disorder dyskeratosis congenita (DC) and in their unaffected relatives (controls). We observed a strong correlation between the Southern blot average TL and the flow FISH total lymphocyte TL in both the DC patients and their unaffected relatives (R2 of 0.68 and 0.73, respectively). The correlation between the qPCR average TL and that of the Southern blot method was modest (R2 of 0.54 in DC patients and of 0.43 in unaffected relatives). Similar results were noted when comparing the qPCR average TL and the flow FISH total lymphocyte TL (R2 of 0.49 in DC patients and of 0.42 in unaffected relatives). In conclusion, the strengths of the correlations between the three widely used TL assays (qPCR, flow FISH, and Southern blot) were significantly different. Careful consideration is warranted when selecting the method of TL measurement for research and for clinical studies

The limitations of qPCR telomere length measurement in diagnosing dyskeratosis congenita

Abstract Background: Telomere length <1st percentile‐for‐age in leukocyte subsets by flow cytometry with fluorescence in situ hybridization (flow FISH) is highly sensitive and specific in diagnosing patients with dyskeratosis congenita (DC), a telomere biology disorder. Methods: We evaluated the clinical utility of the high‐throughput quantitative real‐time PCR (qPCR) relative telomere length (RTL) measurement as a diagnostic test for DC in patients with a priori clinical and/or genetic DC diagnoses. We calculated the sensitivity and specificity of RTL at different age‐specific percentile cutoffs in 31 patients with DC and 51 mutation‐negative relatives, and evaluated RTL difference by disease genotype. Results: qPCR RTL <1st percentile‐for‐age failed to identify more than 60% of the patients already known to have DC (sensitivity = 39%, specificity = 98%). Three‐quarters of DC patients had RTL below the 10th percentile‐for‐age (sensitivity = 74%), as did 12% of the unaffected relatives (specificity = 88%). Conclusions: Our findings suggest that the qPCR RTL method is not optimal for diagnosing DC. In light of these limitations, leukocyte flow FISH telomere length remains the recommended molecular test for diagnosing DC

Correlation of relative telomere length (standardized T/S ratio) of matched subjects across extraction techniques and assay techniques.

<p>Inset heat map displays coefficient of determination (R²) for each correlation.</p