Preparation, characterisation and transcriptome analysis of RNA frm human vCJD brains.
Abstract
The pathological mechanisms of variant Creutzfeldt-Jakob disease (vCJD) in the
human brain remain poorly understood. Gene expression data may provide insight
into the molecular mechanisms involved. This requires analysis of human postmortem
brain tissue however; the variability in RNA preparations from human brain
material is a concern. A method for the isolation of RNA from vCJD brains which
minimized infectivity and reduced Proteinase K resistant prion protein levels to
undetectable by biochemical assay was developed. RNA preparations were made
from sample of the frontal parasagital cortex, sub-frontal cortex and cerebellum of 78
human autopsy cases; 21 vCJD, 26 other neurological disease (OND) and 31 nonneurological
disease (NND).
Suitable RNA metrics for these human brain RNA preparations were evaluated and
the intra- and inter-case variability of RNA preparations was determined. There was
marked intra- and inter-case variability in RNA integrity number (RIN), A260:280
absorbance ratio and RNA yield. In particular, RIN and A260:280 showed little
variation intra-patient, although RNA yield was more variable. The effects of postmortem
interval, tissue pH, age at death, gender, freeze-thaw cycles (including
storage method and temperature) and agonal state were investigated; none of these
parameters correlated with the marked variability observed.
Parameters for matching vCJD and OND/NND cases were considered and RNA
from three age and gender matched comparison groups, each containing one OND,
one NND and one vCJD case, were used for gene expression analysis. Data was
generated using Superarray GEArray® Focused DNA Microarray and analysed using
the GEArray Expression Analysis Suite and Significance Analysis of Microarray
software. A comparison between matched vCJD and NND control cases identified 26
up-regulated and 16 down-regulated genes, showing >1.5-fold change with a false
discovery rate of 9%. The modulated genes were involved in cell signaling, cell
death, cholesterol and lipid metabolism. Involvement of these pathways is consistent
with findings in other transmissible spongiform encephalopathy studies.