Presented at the Neonatal Society 2012 Summer Meeting.
Turner K1, Vasu V1,2, Griffin D1
1 School of Biosciences, University of Kent
2 Department of Neonatal Medicine, William Harvey Hospital
Background: Available data indicate that ex preterm infants manifest an “aged” phenotype characterised by insulin resistance, ectopic adipose tissue distribution and hypertension when compared to term born healthy infants. Telomeres are highly specialised nucleoprotein structures that function to maintain genome stability. It is widely accepted that the shortening of telomeres following each round of replication is directly correlated with ageing. Although several methodologies exist for measuring telomere length, the limitations associated with blood sampling volumes in newborn infants pose a particular challenge for studying telomere biology in this population. We are conducting a study to establish whether preterm at term infants have reduced telomere length when compared to term born healthy infants. Here, we present data comparing traditional telomere restriction fragment (TRF) analysis to a newer quantitative real-time polymerase chain reaction (qRT-PCR) approach to identify a suitable methodology for telomere length analysis in newborn infants. Ethics approval for this study has been granted by Surrey Research Ethics Committee.
Methods: Peripheral blood was collected from adult donors for assay optimisation purposes, and from preterm (<32 weeks gestation) and term (37-42 weeks gestation) infants. Genomic DNA was isolated from 3mls of adult, or 500μl of infant whole blood using the Roche DNA Isolation from Mammalian Blood kit. TRF analysis was carried out using the Roche TeloTAGGG kit. qRT-PCR primers and cycling conditions were based on those described by Cawthon 2009.
Results: Preliminary data using TRF analysis showed large smears that were inconclusive. Furthermore DNA isolation from 500μl newborn infant blood gave a relatively low yield; therefore obtaining triplicate data in TRF analysis was unachievable. To solve this problem qRT-PCR cycling conditions adapted from Cawthon 2009 were optimised.
Conclusion: Low yields of DNA obtained from small sample volumes from infants means that traditional TRF analysis is not practical for accurately testing our hypothesis. Accordingly, we have adapted a published qRT-PCR protocol for sensitive telomere length analysis that requires lower quantities of template material and is less time consuming and labour intense.
Corresponding authors: D.K.Griffin@kent.ac.uk, vimal.vasu@ekht.nhs.uk
References
Cawthon, R.M., 2009. Telomere length measurement by a novel monochrome multiplex quantitative PCR method. Nucleic acids research, 37(3)