Microarray Data Analysis Intensity values of all chips were normalized with dChip software (http://www.dchip.org) [37] and applying the model- based expression analysis algorithm (PM-only or PM/MM model) the expression values were calculated. Global brain gene expression profiles from mice 145 days post- inoculation, with either mock inoculum or the RML prion strain, were compared applying a 1.3-fold change as the lower limit, lower 90% confidence boundary of fold change and p values of ,0.05. Only those genes were considered which were flagged present in 100% of chips from cRNA probes derived from at least either mock or prion inoculated mice. Microarray datasets are available at www. ncbi.nlm.nih.gov/geo; accession number GSE7207. Northern Blot Analyses Northern blot hybridisation experiments as previously described [38] using 15 mg total RNA. Random primed [a32P]dCTP probes were prepared from 20 ng of gel-purified DNA homologous to target transcripts using Rediprime II random priming kit (Amersham Biosciences, Switzerland) and purified with Nick Sephadex G50 columns (Amersham Biosciences, Switzerland). Prehybridization and hybridization was performed overnight at 42uC using Ultrahyb (Ambion, USA), containing 16106 cpm/ml radiolabelled probe and 100 mg/ml denatured herring sperm DNA (Catalysis AG, Switzerland) and 20 mg/ml denatured yeast tRNA (Sigma Fluka, Switzerland). Post-hybridization washes were performed according to Ultrahyb instructions. Hybridization signals were quantified using Fujifilm imaging plate technology (BAS-1800II, Fujifilm, Japan) and normalized for variations in RNA loading by subsequent probing with an excess of 18S rRNA cDNA probe [38]. Probes and accession numbers Murine cDNAs for derivation of probes for Serpin-a3n and Gfap were obtained from Open Biosystems (www.openbiosystems. com); accession numbers were BI145274 and AI836096 respec- tively. Quantitative Real-Time PCR (QPCR) In all cases 5 mg of total RNA was used as template for first- strand cDNA synthesis. Prior to cDNA synthesis, residual genomic DNA was removed by the DNA free-kit (Ambion, USA). Total RNA was converted into cDNA using the bulk first strand cDNA synthesis kit and Not I-(dT)18 as primer (Amersham Biosciences Europe GmbH, Germany). Control reactions omitting reverse transcriptase were performed to validate successful removal of contaminating genomic DNA. Successful cDNA synthesis and lack of contamination with genomic DNA was tested by performing PCR (40 cycles) with primers specific for b-actin. All samples utilized for QPCR analysis were validated free of contaminating genomic DNA. Quantitative real-time PCR was performed using the SYBR Green PCR Master Mix (Qiagen AG, Switzerland) on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Switzerland) using default cycling conditions. For murine samples, triplicate reactions of three biological replicates were utilized. In the case of human material, triplicate reactions were performed for each sample. Expression levels of serpin-a3n/ a1-ACT were normalized to b-actin. Primer sequences were as follows: 59- tatctgcctccacccaaaag-39 & 59- gccagatgtggacaaagtga-39 (murine serpin-a3n), 59-atggatgacgatatcgctg-39 & 59-atgaggtagtctgt- caggt-39 (murine ActB), 59-tctcccaggtggtccataag-39 & 59-ttactga- gagccccactgct-39 (human a1-ACT), 59-ggacttcgagcaagagatgg-39 & 59-agcactgtgttggcgtacag-39 (human ACTB). Prion Disease Biomarker Enzyme-linked immunosorbent assays for a1- antichymotrypsin (a1-ACT) Human a1-ACT was measured by triple antibody sandwich ELISA. Microtiter plate wells (Nunc F96 Maxisorb, Milan Analytica, Switzerland) were coated with 100 ml of 5.4 mg/ml affinity purified rabbit anti-human a1-ACT (Dako, Switzerland) in carbonate-bicarbonate buffer, pH 9.6. The plates were then sealed and incubated overnight at 4uC. After six washes with phosphate- buffered saline (PBS) containing 0.05% Tween-20 (PBST) the remaining binding sites in the wells were blocked by incubating with 100 ml of PBST containing 5% bovine serum albumin (BSA, Sigma) for 2 h at room temperature with shaking, followed by six washes with PBST. Thereafter, 100 ml of purified recombinant human a1-ACT (R&D Systems, UK), in several dilutions ranging from 0 to 200 ng/ml to generate a standard curve, and test samples were added. Standard curve and test samples were diluted into PBS sample buffer containing a final concentration of 0.05% Tween-20 and 1% BSA. Serum, CSF and urine were diluted 1:250 000, 1:3000 and 1:300 respectively such that all samples were contained within the measurable range of the standard curve. Standard curve and test sample dilutions were prepared in triplicate. The plates were sealed and incubated at room temperature for 2 h at room temperature with shaking and washed six times with PBST. Then 100 ml of sheep anti-human a1-ACT (The Binding Site, UK), diluted to 31 mg/ml in PBST containing 1% BSA, was added to each of the wells and the plate sealed and incubated for 2 h at room temperature with shaking. Following a further six washes with PBST 100 ml of horseradish peroxidase (HRP) conjugated donkey anti-sheep IgG, diluted 1:1000 in PBST containing 1% BSA, was added to each well and the plate sealed and incubated at room temperature for 2 h. Following a final six washes with PBST 100 ml of 3,39,5,59- teramethylbenzidine chromogenic substrate (TMB, Invitrogen, UK) was added to each well. Following colour development 100 ml of H2SO4 was added to each well to stop the reaction and the optical density measured at 450 nm or 405 nm in an ELISA micro-plate reader. Values were expressed as concentration per ml fluid (plasma, CSF & urine), relative to total protein (plasma & CSF) or relative to creatinine and albumin (urine). Inter- and intra-assay correlation of variance was 7.36% and 3.84% respectively. Enzyme-linked immunosorbent assays for cystatin C Measurement of human cystatin C was performed using a commercial sandwich ELISA kit (Biovendor Laboratory Medicine Inc., Czech Republic) according to the manufacturer’s instruc- tions. Human cystatin C standard curve and test samples were diluted into PBS sample buffer containing a final concentration of 0.05% Tween-20 and 1% BSA. Serum, cerebrospinal fluid (CSF) and urine were diluted 1:300, 1:1000 and 1:100 respectively such that all assay values were contained within the measurable range of the standard curve. Standard curve and test sample dilutions were prepared in triplicate. Optical density was measured at 450 nm or 405 nm in an ELISA micro-plate reader. Values were expressed as concentration per ml fluid (plasma, CSF & urine), relative to total protein (plasma & CSF) or relative to creatinine and albumin (urine). Albumin & a1-microglobulin Measurements of human urinary albumin and a1-microglobulin were performed using a commercial competitive ELISA for albumin (Orgentec Diagnostika, Germany) and a sandwich ELISA format for a1-microglobulin (ImmunDiagnostik, Germany) ac- PLoS ONE | www.plosone.org 11 December 2008 | Volume 3 | Issue 12 | e3870