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Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer.

ABSTRACT: Excess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR methods analyze the relative length of telomeres by amplifying telomere sequence products and normalizing with single-copy gene products. However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA. Because of genomic instability in cancer cells, we hypothesized that the use of single-copy genes (scg) is less accurate for normalizing data in cancer DNA and that new primer sets are required to better represent relative telomere length in cancer DNA. We first confirmed that cancer cells had a different copy ratio among different scg, implying that DNA is aneuploid. By using the new primer sets that amplify multiple-copy sequences (mcs) throughout the genome, the telomere qPCR results showed that the mcs primers were interchangeable with the scg primers as reference primers in normal DNA. By comparing results from the traditional southern blotting method (as kilobases) and results from monochrome multiplex qPCR using the mcs primers (as T/M ratios), we verified that the T/M ratio is highly correlated with absolute telomere length from the southern blot analysis. Together, the mcs primers were able to represent the telomere lengths accurately in cancer DNA samples. These results would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer.

SUBMITTER: Dahlgren PN

PROVIDER: S-EPMC5915991 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Use of telomere length (TL) as a biomarker for various environmental exposures and diseases has increased in recent years. Various methods have been developed to measure telomere length. Polymerase chain reaction (PCR)-based methods remain wide-spread for population-based studies due to the high-throughput capability. While several studies have evaluated the repeatability and reproducibility of different TL measurement methods, the results have been variable. We conducted a literature review of TL measurement cross-method comparison studies that included a PCR-based method published between January 1, 2002 and May 25, 2020. A total of 25 articles were found that matched the inclusion criteria. Papers were reviewed for quality of methodologic reporting of sample and DNA quality, PCR assay characteristics, sample blinding, and analytic approaches to determine final TL. Overall, methodologic reporting was low as assessed by two different reporting guidelines for qPCR-based TL measurement. There was a wide range in the reported correlation between methods (as assessed by Pearson's r) and few studies utilized the recommended intra-class correlation coefficient (ICC) for assessment of assay repeatability and methodologic comparisons. The sample size for nearly all studies was less than 100, raising concerns about statistical power. Overall, this review found that the current literature on the relation between TL measurement methods is lacking in validity and scientific rigor. In light of these findings, we present reporting guidelines for PCR-based TL measurement methods and results of analyses of the effect of assay repeatability (ICC) on statistical power of cross-sectional and longitudinal studies. Additional cross-laboratory studies with rigorous methodologic and statistical reporting, adequate sample size, and blinding are essential to accurately determine assay repeatability and replicability as well as the relation between TL measurement methods.

Project description:Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R(2) = 0.60; p<0.0001) and patients (R(2) = 0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R(2) = 0.35; p<0.0001) and low correlation for patients (R(2) = 0.20; p = 0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R(2) = 0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R(2) = 0.1, p = 0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8 ± 7.1%) and qPCR (9.5 ± 7.4%; p = 0.35), but the inter-assay CV was lower for flow-FISH (9.6 ± 7.6% vs. 16 ± 19.5%; p = 0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human leukocyte's telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes.

Project description:Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for differences in RTL measurements from samples extracted by different DNA extraction methods within a study.

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Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer.

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