Preparation, characterisation and transcriptome analysis of RNA frm human vCJD brains.
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.