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 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 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 same samples after 6 months at various concentrations and storage temperature conditions.

<p>(a) 1 ng/uL at 4°C, (b) 1 ng/uL at -30°C, (c) 25 ng/uL at 4°C, and (d) 25 ng/uL at -30°C.</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 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

Genetic Susceptibility Loci, Pesticide Exposure and Prostate Cancer Risk

<div><p>Uncovering SNP (single nucleotide polymorphisms)-environment interactions can generate new hypotheses about the function of poorly characterized genetic variants and environmental factors, like pesticides. We evaluated SNP-environment interactions between 30 confirmed prostate cancer susceptibility loci and 45 pesticides and prostate cancer risk in 776 cases and 1,444 controls in the Agricultural Health Study. We used unconditional logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Multiplicative SNP-pesticide interactions were calculated using a likelihood ratio test. After correction for multiple tests using the False Discovery Rate method, two interactions remained noteworthy. Among men carrying two T alleles at rs2710647 in EH domain binding protein 1 (<i>EHBP1</i>) SNP, the risk of prostate cancer in those with high malathion use was 3.43 times those with no use (95% CI: 1.44–8.15) (P-interaction  = 0.003). Among men carrying two A alleles at rs7679673 in <i>TET2</i>, the risk of prostate cancer associated with high aldrin use was 3.67 times those with no use (95% CI: 1.43, 9.41) (P-interaction  = 0.006). In contrast, associations were null for other genotypes. Although additional studies are needed and the exact mechanisms are unknown, this study suggests known genetic susceptibility loci may modify the risk between pesticide use and prostate cancer.</p> </div

Observed and expected distributions for the P values of linear trend for 3,985 tag-SNPs in 230 gene regions related to immune function in a case-control study of PTC risk.

<p>Observed and expected distributions for the P values of linear trend for 3,985 tag-SNPs in 230 gene regions related to immune function in a case-control study of PTC risk.</p

Stratified odds ratios and 95% CI, adjusted for age and state, for associations between pesticides and prostate cancer.

*<p>Noteworthy at an FDR  = 0.20 level.</p

Risk of prostate cancer in the AHS for previously reported susceptibility loci identified from genome wide association studies of prostate cancer.

<p>Single Nucleotide Polymorphism (SNP); Odds Ratio (OR); Confidence Interval (CI); Agricultural Health Study (AHS).</p>*<p>Risk Allele Frequency (RAF) among controls. ** OR per risk allele assuming a log-additive model. Adjusted for age and state.</p>†<p>Completion rate <90%.</p><p>rs600173-rs9623117 r² = 1.0, rs13264338-rs2928679 r² = 0.96.</p