Method specific calibration corrects for DNA extraction method effects on relative telomere length measurements by quantitative PCR

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

Longitudinal changes in telomere length and associated genetic parameters in dairy cattle analysed using random regression models

Telomeres cap the ends of linear chromosomes and shorten with age in many organisms. In humans short telomeres have been linked to morbidity and mortality. With the accumulation of longitudinal datasets the focus shifts from investigating telomere length (TL) to exploring TL change within individuals over time. Some studies indicate that the speed of telomere attrition is predictive of future disease. The objectives of the present study were to 1) characterize the change in bovine relative leukocyte TL (RLTL) across the lifetime in Holstein Friesian dairy cattle, 2) estimate genetic parameters of RLTL over time and 3) investigate the association of differences in individual RLTL profiles with productive lifespan. RLTL measurements were analysed using Legendre polynomials in a random regression model to describe TL profiles and genetic variance over age. The analyses were based on 1,328 repeated RLTL measurements of 308 female Holstein Friesian dairy cattle. A quadratic Legendre polynomial was fitted to the fixed effect of age in months and to the random effect of the animal identity. Changes in RLTL, heritability and within-trait genetic correlation along the age trajectory were calculated and illustrated. At a population level, the relationship between RLTL and age was described by a positive quadratic function. Individuals varied significantly regarding the direction and amount of RLTL change over life. The heritability of RLTL ranged from 0.36 to 0.47 (SE = 0.05–0.08) and remained statistically unchanged over time. The genetic correlation of RLTL at birth with measurements later in life decreased with the time interval between samplings from near unity to 0.69, indicating that TL later in life might be regulated by different genes than TL early in life. Even though animals differed in their RLTL profiles significantly, those differences were not correlated with productive lifespan (p = 0.954)

DNA integrity gels.

<p>(A) Illustrative DNA Integrity gels with gel scores. Example integrity gels for (B) Holstein Friesian cattle and (C) Soay sheep. Individual samples (represented by numbers in image) that were extracted with different DNA extraction protocols. (PG: Gentra Puregene kit, SC: DNeasy spin columns, SP: DNeasy 96 well plate; GS: calibrator DNA (“golden sample”).</p

Raw RTL and Cq values.

<p>RTL or Cq values by DNA extraction method and qPCR plate for cattle (A-E) and sheep (F-I). RTL calculated with method specific (MS) calibrator (A + F), Puregene (PG) calibrator (B), no calibrator (C+G). Cq values for telomere reaction (D+H) and control gene <i>B2M</i> (E+I). Colours represent DNA extraction methods. White: Gentra Puregene, blue: DNeasy spin columns, orange: DNeasy 96 well plate.</p

Variance component and parameter estimates.

<p>Variance component and parameter estimates.</p

Correlations between methods.

<p>Correlations between RTL measurements from different DNA extraction methods (PG: Gentra Puregene kit; SC: DNeasy spin columns; SP: DNeasy 96 well plate): Cattle, method-specific calibrator (A); Cattle, Puregene calibrator (B); Cattle, no calibrator (C); Sheep, method-specific calibrator (D); Sheep, no calibrator (E). Regression lines and their 95% confidence interval are shown in blue and grey, respectively, with red lines reflecting a hypothetically perfect correspondence (slope of one, intercept of zero).</p

Brain matters: unveiling the distinct contributions of region, age, and sex to glia diversity and CNS function

Abstract The myelinated white matter tracts of the central nervous system (CNS) are essential for fast transmission of electrical impulses and are often differentially affected in human neurodegenerative diseases across CNS region, age and sex. We hypothesize that this selective vulnerability is underpinned by physiological variation in white matter glia. Using single nucleus RNA sequencing of human post-mortem white matter samples from the brain, cerebellum and spinal cord and subsequent tissue-based validation we found substantial glial heterogeneity with tissue region: we identified region-specific oligodendrocyte precursor cells (OPCs) that retain developmental origin markers into adulthood, distinguishing them from mouse OPCs. Region-specific OPCs give rise to similar oligodendrocyte populations, however spinal cord oligodendrocytes exhibit markers such as SKAP2 which are associated with increased myelin production and we found a spinal cord selective population particularly equipped for producing long and thick myelin sheaths based on the expression of genes/proteins such as HCN2. Spinal cord microglia exhibit a more activated phenotype compared to brain microglia, suggesting that the spinal cord is a more pro-inflammatory environment, a difference that intensifies with age. Astrocyte gene expression correlates strongly with CNS region, however, astrocytes do not show a more activated state with region or age. Across all glia, sex differences are subtle but the consistent increased expression of protein-folding genes in male donors hints at pathways that may contribute to sex differences in disease susceptibility. These findings are essential to consider for understanding selective CNS pathologies and developing tailored therapeutic strategies

Fixed curve of logarithmically transformed relative leukocyte telomere (RLTL) data.

<p>Blue line: quadratic Legendre polynomial function of age; black solid line: phenotypic RLTL measurements for each month.</p