Subjects
Mouse tissue was provided by the William Harvey Institute, details of husbandry and culling, including compliance with UK Home Office and local ethical guidelines issued by the MRC has been previously published. In short, strains of C57BL/6J mice (10 Nnt+/+, 10 NntBAC and 9 Nnt-/- ), were reared until 18 months of age and terminally anaesthetised via isoflurane. Their tissue was immediately harvested and flash frozen 21.
In our lab., gDNA was extracted from kidney tissue from all mice (n=29) and from liver tissue of the wildtype strain (n=9). The relative telomere length of each sample was then determined by mmQPCR.
DNA Extraction
For each individual, gDNA was extracted in one of three ways. 1. Traditional phenol/chloroform followed by ethanol precipitation. Briefly, 1cm3 of liver tissue was digested overnight in a 37°C water bath in a 100µl solution consisting of 0.1MNaCl, 10nM Tris-base, 1mM EDTA, 1%SDS and 2µl proteinase K(All reagents supplied from Sigma Aldrich). Following digestion an equal volume of Phenol-Chloroform-isoamyl alcohol (Sigma Aldrich) was added. Samples were vortexed and spun at high speed in a centrifuge. Following separation, the aqueous phase was removed and added to a fresh tube. Pure gDNA was then extracted from the aqueous phase utilising standard ethanol precipitation procedure. 2. A magnetic bead-based method (MagAttract HMW DNA Kit, Qiagen Ltd, Manchester UK). 3. Spindown columns (PureLinkTM Invitrogen, Carlsbad, CA, USA). Fro (2) and (3) DNA was extracted using the kits as per the manufacturer’s recommendations. Following extraction, purities and yields were analysed via NanoDrop. For each sample, aliquots at a concentration of 10ng/µl were produced in preparation for telomere length estimation.
T/S ratio determination
Following extraction, relative telomere length was determined by mmQPCR, as previously described with some modifications. This is a method which multiplexes the PCR and enables quantification of the target and the single copy gene within the same qPCR reaction and has been shown to reduce experimental error 15. The SCG 36b4 was selected as it is highly conserved in mice and has been previously used to investigate murine TL 17. We used TL primers as previously published and modified the published SCG primers, by the addition of 5' GC clamps, in line with Cawthorn et al.,15 thus increasing the tm of the amplicon allowing two discrete amplification profiles (see table 2).
For each plate, standards spanning a range of concentrations from 2.5ng/µl to 40ng/µl were run in triplicate, each sample was analysed in duplicate. Each 10µl reaction consisted of the following reagents: 5µl iQ SYBR green supermix (Biorad), 900nM TelG and TelC primers 500nM 36B4GCF and 36B4GCR primers (all supplied from Eurogentec) [Table 1], 10ng of gDNA and 1.2µl dH20. The mmQPCR reaction was carried out using 96-well plates on an Agilent AriaMx thermocycler. The cycling conditions were as follows (15mins @ 95°C) x 1, (15s @ 94°C, 15s @49°C) x 2, (15s @ 94°C, 10s @ 62°C, 15S @ 74°C with signal acquisition, 10s @ 84°C, 15s @ 88°C with signal acquisition) x 30. Following amplification, all samples were subject to high resolution melt curve analysis to check the specificity of the reaction.
Statistics And Reproducibility
All analysis was conducted in SPSS(v23.0). A t-test (ANOVA) was utilised to compare the differences in the resultant T/S ratios from each extraction method. Results with p <0.05 were deemed statistically significant. Graphs were generated using GraphPad Prism (v7.02).
For each plate, standards spanning a range of concentrations from 2.5ng/µl to 40ng/µl were run in triplicate, each sample was analysed in duplicate. Any pair of samples which generated a T/S ratio with a coefficient of variation greater than 30% were deemed inaccurate and were repeated.