Preece, Paul Simon; (2004) Quantification of gene expression in post-mortem human brain. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Quantitative human mRNA data are derived from post-mortem or biopsied tissue. Confounding factors, RNA degradation, poor replication and a large variance are often cited, however, as objections to the data's reliability. At issue is whether post-mortem mRNA represents an ordered system and to what degree non-specific factors contribute to the measurements. I developed statistical methods and validated them by measuring 25 mRNA transcripts in an animal model of ischaemia. In the process I discovered novel increases for 3 genes in rats with ischaemic damage: leukaemia inhibitory factor, nestin and galanin mRNA. Additionally, I discovered that reference genes known as "housekeepers"' do not always act as steady-state controls and that the precise value of a test gene response varies according to the baseline choice of reference gene. Once optimised, I applied the analytical methods to human post-mortem brains. I used TaqMan™ real-time RT-PCR to measure 13 mRNAs in 513 cortical samples taken from 90 Alzheimer's disease and 81 control brains. Despite a high variance and confounding factors such as brain pH, I found strong geometric relations between the mRNA transcripts up to and beyond 100 hours autopsy delay. Where a postmortem brain had a high/low level of one mRNA, the same brain invariably had a high/low level of other mRNAs; correlated order is present and provides a means of isolating any mRNA change due specifically to disease. I measured mRNA levels of β-Secretase (BACE), GSK 3 and the isoforms of APP/APRP in the AD and control brains. After adjustment for age of death, brain pH, and gender, there was no change in the mRNA levels for either BACE or GSK 3α mRNA (p = 0.354 and p = 0.054 respectively). There was a change, however, in the ratio of KPI+ to KPI- mRNA isoforms of APP/APRP. Three separate probes, designed only to recognise KPI+ mRNA, each gave increases of between 28 and 50% in AD brains relative to controls (p = 0.002). There was no change in the mRNA levels of KPI-(APP 695) (p = 0.898). Therefore, whilst I KPI- mRNA levels remained level between AD and control brains, the KPI+ species were seen to increase specifically in the AD brains.

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