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1.  Blood and Dried Blood Spot Telomere Length Measurement by qPCR: Assay Considerations 
PLoS ONE  2013;8(2):e57787.
Measurement of telomere length is crucial for the study of telomere maintenance and its role in molecular pathophysiology of diseases and in aging. Several methods are used to measure telomere length, the choice of which usually depends on the type and size of sample to be assayed, as well as cost and throughput considerations. The goal of this study was to investigate the factors that may influence the reliability of qPCR-based relative telomere length measurements in whole blood. Day to day intra-individual variability, types of blood anticoagulant, sample storage conditions, processing and site of blood draw were investigated. Two qPCR-based methods to measure telomere length (monoplex vs. multiplex) were also investigated and showed a strong correlation between them. Freezing and thawing of the blood and storage of the blood at 4°C for up to 4 days did not affect telomere length values. Telomere lengths in dried blood spots were significantly higher than both whole blood and peripheral mononuclear blood cells, and were highly correlated with both. We found that telomere length measurements were significantly higher in dried blood spots collected directly from fingertip prick compared to dried blood spots prepared with anticoagulated whole blood collected from the finger, and non-blotted whole blood taken from both finger and arm venipuncture. This suggests that DNA from cells blotted on paper is not equivalent to that collected from venipuncture whole blood, and caution should be taken when comparing between blood sample types.
PMCID: PMC3581490  PMID: 23451268
2.  Telomere length measurement by a novel monochrome multiplex quantitative PCR method 
Nucleic Acids Research  2009;37(3):e21.
The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction wells, and single copy gene (S) signals in separate wells, in comparison to a reference DNA, to yield relative T/S ratios that are proportional to average telomere length. Multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed. Here, we present the first multiplexed QPCR method for telomere length measurement. Remarkably, a single fluorescent DNA-intercalating dye is sufficient in this system, because T signals can be collected in early cycles, before S signals rise above baseline, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation of T/S ratios with Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this monochrome multiplex QPCR method (R2 = 0.844) than with our original singleplex method (R2 = 0.677). Multiplex T/S results from independent runs on different days were highly reproducible (R2 = 0.91).
PMCID: PMC2647324  PMID: 19129229
3.  Impartial comparative analysis of measurement of leukocyte telomere length/DNA content by Southern blots and qPCR 
Nucleic Acids Research  2011;39(20):e134.
Telomere length/DNA content has been measured in epidemiological/clinical settings with the goal of testing a host of hypotheses related to the biology of human aging, but often the conclusions of these studies have been inconsistent. These inconsistencies may stem from various reasons, including the use of different telomere length measurement techniques. Here, we report the first impartial evaluation of measurements of leukocyte telomere length by Southern blot of the terminal restriction fragments and quantitative PCR (qPCR) of telomere DNA content, expressed as the ratio of telomeric product (T)/single copy gene (S) product. Blind measurements on the same samples from 50 donors were performed in two independent laboratories on two different occasions. Both the qPCR and Southern blots displayed highly reproducible results as shown by r values > 0.9 for the correlations between results obtained by either method on two occasions. The inter-assay CV measurement for the qPCR was 6.45%, while that of the Southern blots was 1.74%. The relation between the results generated by Southern blots versus those generated by qPCR deviated from linearity. We discuss the ramifications of these findings with regard to measurements of telomere length/DNA content in epidemiological/clinical circumstances.
PMCID: PMC3203599  PMID: 21824912
4.  Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods 
BMC Genetics  2012;13:77.
Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay.
Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays.
Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.
PMCID: PMC3489520  PMID: 22954451
Quantitative PCR; Telomere length; Quality control; Non-model species; Guidelines
5.  Associations of leukocyte telomere length with body anthropometric indices and weight change in Chinese women 
Obesity (Silver Spring, Md.)  2013;21(12):10.1002/oby.20321.
Telomeres are specialized chromatin structures essential for maintenance of chromosomal integrity and stability. Obesity has been proposed to be associated with telomere shortening; however, epidemiologic evidence has been conflicting. We conducted a study to evaluate the associations of telomere length with various anthropometric indices of general and abdominal obesity, as well as weight change.
Design and Methods
The study included 2,912 Chinese women ages 40–70 years. Monochrome multiplex quantitative PCR was applied to measure relative telomere length. ANOVA and the Dunnett test were used to compare log-transformed relative telomere length. Tests for linear trend were performed by entering the ordinal exposure as continuous parameters in the models.
There is an inverse association between telomere length and body mass index (BMI) (Ptrend = 0.005), waist circumference (Ptrend = 0.004), waist-to-height ratio (WHtR) (Ptrend = 0.004), weight (Ptrend = 0.010), and hip circumference (Ptrend = 0.026), but not waist-to-hip ratio (WHR) (Ptrend = 0.116) or height (Ptrend = 0.675). Weight change since age 50 was further evaluated among women over age 55. Women who maintained their weight within ±5% since age 50, particularly within a normal range (BMI = 18.5–24.9 kg/m2), or reduced their weight from overweight (BMI = 25–29.9 kg/m2) or obesity (BMI ≥30 kg/m2) to normal range, had a longer mean of current telomere length than women who gained weight since age 50 (Ptrend = 0.025), particularly those who stayed in obesity or gained weight from normal range or overweight to obesity (P = 0.023).
Our findings provide strong evidence supporting the hypothesis that telomere shortening is associated with obesity and that maintaining body weight within a normal range helps maintain telomere length.
PMCID: PMC3676725  PMID: 23408544
6.  Multiplex qPCR for Detection and Absolute Quantification of Malaria 
PLoS ONE  2013;8(8):e71539.
We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R2 values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.
PMCID: PMC3756973  PMID: 24009663
7.  Reliability and Short-Term Intra-Individual Variability of Telomere Length Measurement Using Monochrome Multiplexing Quantitative PCR 
PLoS ONE  2011;6(9):e25774.
Studies examining the association between telomere length and cancer risk have often relied on measurement of telomere length from a single blood draw using a real-time PCR technique. We examined the reliability of telomere length measurement using sequential samples collected over a 9-month period.
Methods and Findings
Relative telomere length in peripheral blood was estimated using a single tube monochrome multiplex quantitative PCR assay in blood DNA samples from 27 non-pregnant adult women (aged 35 to 74 years) collected in 7 visits over a 9-month period. A linear mixed model was used to estimate the components of variance for telomere length measurements attributed to variation among women and variation between time points within women. Mean telomere length measurement at any single visit was not significantly different from the average of 7 visits. Plates had a significant systematic influence on telomere length measurements, although measurements between different plates were highly correlated. After controlling for plate effects, 64% of the remaining variance was estimated to be accounted for by variance due to subject. Variance explained by time of visit within a subject was minor, contributing 5% of the remaining variance.
Our data demonstrate good short-term reliability of telomere length measurement using blood from a single draw. However, the existence of technical variability, particularly plate effects, reinforces the need for technical replicates and balancing of case and control samples across plates.
PMCID: PMC3184167  PMID: 21984947
8.  Telomere length in blood and skeletal muscle in relation to measures of glycaemia and insulinaemia 
Skeletal muscle is a major metabolic organ and plays important roles in glucose metabolism, insulin sensitivity, and insulin action. Muscle telomere length reflects the myocyte's exposure to harmful environmental factors. Leukocyte telomere length is considered a marker of muscle telomere length and is used in epidemiologic studies to assess associations with ageing-related diseases where muscle physiology is important. However, the extent to which leucocyte telomere length and muscle telomere length are correlated is unknown, as are their relative correlations with glucose and insulin concentrations. The purpose of this study was to determine the extent of these relationships.
Leucocyte telomere length and muscle telomere length were measured by quantitative real-time PCR in participants from the Malmö Exercise Intervention (MEI; n=27) and the PPP-Botnia studies (n=31). Participants in both studies were free from type 2 diabetes. We assessed the association between leucocyte telomere length, muscle telomere length and metabolic traits using Spearmen correlations and multivariate linear regression. Bland-Altman analysis was used to assess agreement between leucocyte telomere length and muscle telomere length.
In age-, study-, diabetes family history- and sex-adjusted models, leucocyte telomere length and muscle telomere length were positively correlated (r=0.39, 95% CI: 0.15, 0.59). Leucocyte telomere length was inversely associated with 2hr glucose concentrations (r= -0.58, 95% CI: -1.0, -0.16), but there was no correlation between muscle telomere length and 2 hr glucose concentrations (r=0.05, 95% CI: -0.35, 0.46) or between leucocyte telomere length or muscle telomere length with other metabolic traits.
In summary, the current study supports the use of leucocyte telomere length as a proxy for muscle telomere length in epidemiological studies of type 2 diabetes aetiology.
PMCID: PMC3698879  PMID: 22747879
Leukocyte telomere length; muscle telomere length; cardiometabolic; type 2 diabetes; skeletal muscle physiology
9.  Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens 
Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens.
The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%.
The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.
PMCID: PMC1852093  PMID: 17407544
10.  A Prospective Study of Telomere Length Measured by Monochrome Multiplex Quantitative PCR and Risk of Lung Cancer 
Telomere length plays an important role in chromosomal stability and tumorigenesis, and its measurement in peripheral white blood cell DNA may be a predictor of the development of lung cancer.
Experimental Design
Using a new method - monochrome multiplex quantitative PCR -which reduces measurement variability, we compared telomere length relative to standard DNA in white blood cell DNA in 229 incident male lung cancer cases and 229 matched controls within the prospective Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of male smokers.
Median (10th, 90th percentile) telomere length was 1.13 (0.86, 1.45) in cases and 1.08 (0.85, 1.38) in controls (P = 0.038). Telomere length was inversely associated with pack-years of smoking (Spearman correlation r = −0.16, P = 0.02) among controls. Compared to subjects with shorter telomere length (≤ median), subjects with greater telomere length (> median) had a 1.6-fold (95% CI, 1.06–2.36) increased risk of lung cancer. There was a significant linear relationship between quartiles of telomere length and risk of lung cancer (odds ratios (95% confidence intervals) by quartile: 1.00, 0.98 (0.55–1.73), 1.62 (0.95–2.77), and 1.50 (0.84–2.68); Ptrend = 0.05). In addition, subgroup analysis showed that greater telomere length was associated with increased risk of lung cancer among heavy smokers (> 38 years) (OR, 1.90; 95% CI, 1.00–3.59) but not among light smokers (≤ 38 years) (OR, 1.08; 95% CI, 0.56–2.11) (Pinteraction = 0.01).
Our results suggest that greater telomere length may be associated with higher risk of lung cancer among male smokers.
PMCID: PMC3509808  PMID: 21507503
Telomere length; lung cancer; cohort study
11.  Association of leukocyte telomere length with colorectal cancer risk: nested case-control findings from the Shanghai Women's Health Study 
Telomeres are specialized chromatin structures essential for maintenance of chromosomal integrity and stability. Abnormal alteration of telomere length has been linked to several cancers; however, epidemiologic evidence regarding the association of telomere length with colorectal cancer risk has been conflicting.
We conducted a nested case-control study to evaluate the association between telomere length and colorectal cancer risk using peripheral blood samples collected prior to cancer diagnosis. The study included 441 women with incident colorectal cancer and 549 matched controls. Monochrome multiplex quantitative PCR was applied to measure relative telomere length. Multiple logistic regressions were used to derive adjusted odds ratios (OR) with 95% confidence intervals (CI) as the measure of association between telomere length and subsequent colorectal cancer risk.
A U-shaped association was observed between telomere length and colorectal cancer risk (test for nonlinearity P = 0.0112). Women with telomere length in the third quintile (40th to 60th percentiles) had the lowest risk of colorectal cancer, and the risks were elevated with a shorter or longer telomere length. This U-shaped association did not statistically differ for colon cancer and rectum cancer.
Conclusions and Impact
Our prospective study revealed a U-shaped association between telomere length in peripheral blood cells and colorectal cancer risk. Our findings provide strong evidence that both very short and very long telomeres are associated with increased risk of colorectal cancer.
PMCID: PMC3467322  PMID: 22911335
12.  Analysis of telomere length in couples experiencing idiopathic recurrent pregnancy loss 
Telomere length plays a significant role in various disorders; however, its role in idiopathic recurrent pregnancy loss (iRPL) is not known. The objective of this study was to assess telomere length in peripheral blood leukocytes in couples experiencing unexplained recurrent pregnancy loss (iRPL).
The study included 25 couples experiencing iRPL and 20 controls. The mean relative telomere length was measured by quantitative Real Time PCR (Q-PCR) based assay, which measures the average ratio of telomere repeat copy number to a single copy gene (36B4) copy number (T/S ratio) in each sample.
The relative leukocyte mean telomere length (T/S) in both men and women from iRPL group was significantly lower (p < 0.05) when compared to controls. A significant (P < 0.05) negative correlation was found between age and leukocyte telomere length (T/S ratio). Among the sperm parameters seminal volume was found to be negatively (r = −0.4679) associated with the telomere T/S ratio. The DNA fragmentation index of sperm showed positive correlation (r = 0.4744) with telomere length. In this preliminary study, we found that shorter telomere length in both men and women may be associated with early pregnancy loss.
In conclusion, shorter telomere length in both male and female partners appears to play a role in the idiopathic recurrent pregnancy loss. Loss of telomeric DNA due to oxidative stress needs further analysis. Analysis of telomere length in germ cells are needed to further substantiate the findings of this study.
PMCID: PMC3696449  PMID: 23608779
Telomere length; Recurrent pregnancy loss; Q-PCR, Sperm chromatin structure assay; Reactive oxygen species
13.  Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay 
PLoS ONE  2011;6(12):e29101.
Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.
PMCID: PMC3237602  PMID: 22194997
14.  A prospective study of telomere length measured by monochrome multiplex quantitative PCR and risk of non-Hodgkin lymphoma 
Telomere length plays an important role in maintaining chromosomal stability and in tumorigenesis. We hypothesized that telomere length in peripheral white blood cell DNA obtained from healthy individuals would be a predictor of future risk of developing non-Hodgkin lymphoma (NHL).
Experimental Design
Using a new assay to measure relative telomere length, monochrome multiplex quantitative PCR, which strongly correlates with telomere length measured by Southern blot (Spearman r = 0.91, p < 0.0001) and has high precision (coefficient of variation = 7%), we compared telomere length in peripheral white blood cell DNA in 107 incident male NHL cases and 107 matched controls within the prospective Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study cohort.
Median (10th, 90th percentile) telomere length was 1.10 (0.79, 1.43) in cases and 1.02 (0.78, 1.26) in controls (p = 0.0017, Wilcoxon sign test). There was a strong dose-response relationship between quartiles of telomere length and risk of NHL overall [odds ratios (95% confidence intervals) by quartile: 1.0; 1.1 (0.4-2.7); 1.8 (0.7-4.9); and 3.6 (1.4-8.9); p trend = 0.003)], and this association was similar across the most common NHL subtypes present in this study.
These results suggest that longer telomere length may be a potential predictor for future risk of NHL.
PMCID: PMC2787641  PMID: 19934287
Telomere length; non-Hodgkin lymphoma; cohort
15.  Telomere length in peripheral blood and breast cancer risk in a prospective case-cohort analysis: results from the Sister Study 
Cancer causes & control : CCC  2011;22(7):1061-1066.
Telomeres are required for maintaining genomic integrity and may play a role in carcinogenesis. Some, but not all, epidemiologic studies have found that short telomeres in leukocytes are associated with an increased risk of breast cancer. To further elucidate this potential association, we examined telomere length in relation to breast cancer risk in prospectively collected blood samples from the Sister Study, a cohort of women aged 35-74 years who have a sister with breast cancer.
We performed a case-cohort analysis comparing incident breast cancer cases (n=342) with a subcohort (n=735), randomly selected from 29,026 participants enrolled by June 1, 2007. Relative telomere length in peripheral blood cells was estimated using a single tube monochrome multiplex quantitative PCR assay.
No association was observed between telomere length and breast cancer risk. Compared to the longest quartile, hazard ratios (HR) associated with the second, third and the shortest quartile were 0.91 (95% confidence interval [95% CI]: 0.62-1.34), 1.11 (95% CI: 0.77-1.60) and 0.93 (95% CI: 0.64-1.35), respectively. Subgroup analyses by menopausal status, invasiveness or estrogen-receptor status of breast cancer did not reveal evidence of association between telomere length in blood cells and subsequent breast cancer risk.
This prospective investigation does not support telomere length in blood cells as a biomarker for breast cancer risk.
PMCID: PMC3445257  PMID: 21643930
breast cancer; telomere length; prospective study; biomarker; qPCR
16.  Correlation of Chromosomal Instability, Telomere Length and Telomere Maintenance in Microsatellite Stable Rectal Cancer: A Molecular Subclass of Rectal Cancer 
PLoS ONE  2013;8(11):e80015.
Colorectal cancer (CRC) tumor DNA is characterized by chromosomal damage termed chromosomal instability (CIN) and excessively shortened telomeres. Up to 80% of CRC is microsatellite stable (MSS) and is historically considered to be chromosomally unstable (CIN+). However, tumor phenotyping depicts some MSS CRC with little or no genetic changes, thus being chromosomally stable (CIN-). MSS CIN- tumors have not been assessed for telomere attrition.
Experimental Design
MSS rectal cancers from patients ≤50 years old with Stage II (B2 or higher) or Stage III disease were assessed for CIN, telomere length and telomere maintenance mechanism (telomerase activation [TA]; alternative lengthening of telomeres [ALT]). Relative telomere length was measured by qPCR in somatic epithelial and cancer DNA. TA was measured with the TRAPeze assay, and tumors were evaluated for the presence of C-circles indicative of ALT. p53 mutation status was assessed in all available samples. DNA copy number changes were evaluated with Spectral Genomics aCGH.
Tumors were classified as chromosomally stable (CIN-) and chromosomally instable (CIN+) by degree of DNA copy number changes. CIN- tumors (35%; n=6) had fewer copy number changes (<17% of their clones with DNA copy number changes) than CIN+ tumors (65%; n=13) which had high levels of copy number changes in 20% to 49% of clones. Telomere lengths were longer in CIN- compared to CIN+ tumors (p=0.0066) and in those in which telomerase was not activated (p=0.004). Tumors exhibiting activation of telomerase had shorter tumor telomeres (p=0.0040); and tended to be CIN+ (p=0.0949).
MSS rectal cancer appears to represent a heterogeneous group of tumors that may be categorized both on the basis of CIN status and telomere maintenance mechanism. MSS CIN- rectal cancers appear to have longer telomeres than those of MSS CIN+ rectal cancers and to utilize ALT rather than activation of telomerase.
PMCID: PMC3836975  PMID: 24278232
17.  The Relationship between Telomere Length and Mortality in Chronic Obstructive Pulmonary Disease (COPD) 
PLoS ONE  2012;7(4):e35567.
Some have suggested that chronic obstructive pulmonary disease (COPD) is a disease of accelerated aging. Aging is characterized by shortening of telomeres. The relationship of telomere length to important clinical outcomes such as mortality, disease progression and cancer in COPD is unknown. Using quantitative polymerase chain reaction (qPCR), we measured telomere length of peripheral leukocytes in 4,271 subjects with mild to moderate COPD who participated in the Lung Health Study (LHS). The subjects were followed for approximately 7.5 years during which time their vital status, FEV1 and smoking status were ascertained. Using multiple regression methods, we determined the relationship of telomere length to cancer and total mortality in these subjects. We also measured telomere length in healthy “mid-life” volunteers and patients with more severe COPD. The LHS subjects had significantly shorter telomeres than those of healthy “mid-life” volunteers (p<.001). Compared to individuals in the 4th quartile of relative telomere length (i.e. longest telomere group), the remaining participants had significantly higher risk of cancer mortality (Hazard ratio, HR, 1.48; p = 0.0324) and total mortality (HR, 1.29; p = 0.0425). Smoking status did not make a significant difference in peripheral blood cells telomere length. In conclusion, COPD patients have short leukocyte telomeres, which are in turn associated increased risk of total and cancer mortality. Accelerated aging is of particular relevance to cancer mortality in COPD.
PMCID: PMC3338848  PMID: 22558169
18.  A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data 
PLoS ONE  2010;5(8):e12355.
Quantitative PCR (qPCR) is a workhorse laboratory technique for measuring the concentration of a target DNA sequence with high accuracy over a wide dynamic range. The gold standard method for estimating DNA concentrations via qPCR is quantification cycle () standard curve quantification, which requires the time- and labor-intensive construction of a standard curve. In theory, the shape of a qPCR data curve can be used to directly quantify DNA concentration by fitting a model to data; however, current empirical model-based quantification methods are not as reliable as standard curve quantification.
Principal Findings
We have developed a two-parameter mass action kinetic model of PCR (MAK2) that can be fitted to qPCR data in order to quantify target concentration from a single qPCR assay. To compare the accuracy of MAK2-fitting to other qPCR quantification methods, we have applied quantification methods to qPCR dilution series data generated in three independent laboratories using different target sequences. Quantification accuracy was assessed by analyzing the reliability of concentration predictions for targets at known concentrations. Our results indicate that quantification by MAK2-fitting is as reliable as standard curve quantification for a variety of DNA targets and a wide range of concentrations.
We anticipate that MAK2 quantification will have a profound effect on the way qPCR experiments are designed and analyzed. In particular, MAK2 enables accurate quantification of portable qPCR assays with limited sample throughput, where construction of a standard curve is impractical.
PMCID: PMC2930010  PMID: 20814578
19.  Telomere Length in Peripheral Blood Leukocytes Is Associated with Risk of Colorectal Cancer in Chinese Population 
PLoS ONE  2014;9(2):e88135.
Human telomeres, tandem repeats of TTAGGG nucleotides at the ends of chromosomes, are essential for maintaining genomic integrity and stability. Results of previous epidemiologic studies about the association of telomere length with risk of colorectal cancer (CRC) have been conflicting.
A case-control study was conducted in a Han population in Wuhan, central China. The relative telomere length (RTL) was measured in peripheral blood leukocytes (PBLs) using quantitative real-time polymerase chain reaction (PCR) in 628 CRC cases and 1,256 age and sex frequency matched cancer-free controls. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using unconditional logistic regression models to evaluate the association between RTL and CRC risk.
Using median RTL in the controls as the cutoff, individuals with shorter RTL were associated with a significantly increased risk of CRC (adjusted OR = 1.27, 95%CI: 1.05–1.55). When participants were further categorized into 3 and 4 groups according to the tertile and quartile RTL values of controls, significant relationships were still observed between shorter RTL and increased CRC risk (OR per tertile = 1.13, 95%CI: 1.00–1.28, Ptrend = 0.045; OR per quartile = 1.12, 95%CI: 1.03–1.23, Ptrend = 0.012). In stratified analyses, significant association between shorter RTL and increased CRC risk was found in females, individuals younger than 60 years old, never smokers and never drinkers.
This study suggested that short telomere length in PBLs was significantly associated with an increased risk of CRC in Chinese Han population. Further validation in large prospective studies and investigation of the biologic mechanisms are warranted.
PMCID: PMC3912164  PMID: 24498432
20.  Telomere Length from Blood Cells and Breast Cancer Risk: Investigations in two case-control studies 
Telomere dysfunction, which leads to genomic instability, is hypothesized to play a causal role in the development of breast cancer. However, the few epidemiologic studies that assessed the relationship between telomere length in blood cells and breast cancer risk have been inconsistent. We conducted two case-control studies to further understand the role of telomere length and breast cancer risk.
Overall telomere lengths were measured by telomere quantitative fluorescent in situ hybridization (TQ-FISH) and telomere quantitative real-time PCR (TQ-PCR). The associations between telomere length from blood leucocytes and risk of breast cancer were examine in two breast cancer case-control studies that were conducted at Roswell Park Cancer Institute (RPCI) and Lombardi Comprehensive Cancer Center (LCCC).
Using the 50th percentile value in controls as a cut point, women who had shorter telomere length were not at significantly increased risk of breast cancer compared with women who had longer telomere length in the RPCI study (odds ratio [OR] = 1.34, 95% confidence interval [CI] = 0.84 to 2.12), in the LCCC study (OR = 1.18, 95% CI = 0.73 to 1.91), or in the combined RPCI and LCCC studies (OR = 1.23, 95% CI = 0.89 to 1.71). There was no significant dose-response relationship across quartiles of telomere length and no significant difference when comparing women in the lowest to highest quartile of telomere length.
Overall telomere length from blood leucocytes was not significantly associated with the risk of breast cancer.
PMCID: PMC3700420  PMID: 19543829
Telomere length; blood leucocytes; breast cancer; biomarkers; genetic susceptibility
21.  STEM-LOOP RT-qPCR for miRNAS 
This unit presents a specific and sensitive quantitative reverse-transcription PCR (RT-qPCR) method for measuring individual microRNAs (miRNAs) in tissue or cultured cells. MiRNAs are 17 – 24 nucleotides (nt) in length. Standard and quantitative PCR methods require a template that is at least twice the length of either of the specific forward or reverse primers, each typically ∼ 20 nt in length. Thus, the target minimum length is ≥ 40 nt, making miRNAs too short for standard RT-qPCR methods. In this assay, each of the RT-qPCR nucleic acid reagents, including the RT-primer, the forward and reverse PCR primers, and the hydrolysis probe, contain design features that, together, optimize miRNA specificity and assay sensitivity. The RT-primer contains a highly stable stem-loop structure that lengthens the target cDNA. The forward PCR primer adds additional length with nucleotides that optimize its melting temperature (Tm) and enhance assay specificity. The reverse primer disrupts the stem loop. Assay specificity is further optimized by placement of the probe over much of the original miRNA sequence, and the probe Tm is optimized by addition of a minor groove binding (MGB) moiety.
PMCID: PMC3152947  PMID: 21732315
22.  Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve 
Nucleic Acids Research  2004;32(6):e56.
Amplification of a cDNA product by quantitative PCR (qPCR) is monitored by a fluorescent signal proportional to the amount of produced amplicon. The qPCR amplification curve usually displays an exponential phase followed by a non-exponential phase, ending with a plateau. Contrary to prevalent interpretation, we demonstrate that under standard qPCR conditions, the plateau can be explained by depletion of the probe through Taq polymerase- catalysed hydrolysis. Knowing the probe concentration and the fluorescence measured at the plateau, a specific fluorescence can thus be calculated. As far as probe hydrolysis quantitatively reflects amplicon synthesis, this, in turn, makes it possible to convert measured fluorescence levels in the exponential phase into concentrations of produced amplicon. It follows that the absolute target cDNA concentration initially engaged in the qPCR can be directly estimated from the fluorescence data, with no need to refer to any calibration with known concentrations of target DNA.
PMCID: PMC390378  PMID: 15054124
23.  Measurement of telomere DNA content by dot blot analysis 
Nucleic Acids Research  2011;39(12):e84.
Telomeres play a central role in human cancer, cardiovascular aging and possibly longevity. However, present methods to measure telomere length are fraught with shortcomings that limit their use. Here, we describe a novel method to measure the relative telomere DNA content by dot blot analysis. In each dot, the DNA content is measured by a DNA stain (Dx) and the telomeric DNA content is measured with a telomeric probe (T). The T normalized for Dx (T/Dx) of each dot is a measure of telomere content. The method requires ∼20 ng of DNA per assay. Moreover, the T/Dx data are highly correlated linearly with mean telomere lengths derived from Southern blots of the terminal restriction fragments (r > 0.96, P < 0.0001). The method is also simple to use, has a relatively low interassay coefficient of variation (<6%), retains its precision in moderately degraded DNA and can be forged for high throughput analysis. The method might help researchers and clinicians alike in understanding risks for and extent of human diseases.
PMCID: PMC3130292  PMID: 21525130
24.  The Association of Telomere Length with Colorectal Cancer Differs by the Age of Cancer Onset 
Telomeres are nucleoprotein structures that cap the end of chromosomes and shorten with sequential cell divisions in normal aging. Short telomeres are also implicated in the incidence of many cancers, but the evidence is not conclusive for colorectal cancer (CRC). Therefore, the aim of this study was to assess the association of CRC and telomere length.
In this case–control study, we measured relative telomere length from peripheral blood leukocytes (PBLs) DNA with quantitative PCR in 598 CRC patients and 2,212 healthy controls.
Multivariate analysis indicated that telomere length was associated with risk for CRC, and this association varied in an age-related manner; younger individuals (≤50 years of age) with longer telomeres (80–99 percentiles) had a 2–6 times higher risk of CRC, while older individuals (>50 years of age) with shortened telomeres (1–10 percentiles) had 2–12 times the risk for CRC. The risk for CRC varies with extremes in telomere length in an age-associated manner.
Younger individuals with longer telomeres or older individuals with shorter telomeres are at higher risk for CRC. These findings indicate that the association of PBL telomere length varies according to the age of cancer onset and that CRC is likely associated with at minimum two different mechanisms of telomere dynamics.
PMCID: PMC3972691  PMID: 24598784
25.  Novel Telomere-Anchored PCR Approach for Studying Sexual Stage Telomeres in Aspergillus nidulans 
PLoS ONE  2014;9(6):e99491.
Telomere length varies between germline and somatic cells of the same organism, leading to the hypothesis that telomeres are lengthened during meiosis. However, little is known about the meiotic telomere length in many organisms. In the filamentous fungus Aspergillus nidulans, the telomere lengths in hyphae and asexual spores are invariant. No study using existing techniques has determined the telomere length of the sexual ascospores due to the relatively low abundance of pure meiotic cells in A. nidulans and the small quantity of DNA present. To address this, we developed a simple and sensitive PCR strategy to measure the telomere length of A. nidulans meiotic cells. This novel technique, termed “telomere-anchored PCR,” measures the length of the telomere on chromosome II-L using a small fraction of the DNA required for the traditional terminal restriction fragment (TRF) Southern analysis. Using this approach, we determined that the A. nidulans ascospore telomere length is virtually identical to telomeres of other cell types from this organism, approximately 110 bp, indicating that a surprisingly strict telomere length regulation exists in the major cell types of A. nidulans. When the hyphal telomeres were measured in a telomerase reverse transcriptase (TERT) knockout strain, small decreases in length were readily detected. Thus, this technique can detect telomeres in relatively rare cell types and is particularly sensitive in measuring exceptionally short telomeres. This rapid and inexpensive telomere-anchored PCR method potentially can be utilized in other filamentous fungi and types of organisms.
PMCID: PMC4057176  PMID: 24927411

Results 1-25 (920502)