Prion Disease Biomarker Figure 6. Western blot analysis of urinary a1-ACT from natural field cases of prion disease. Equivalent amounts of urinary protein from natural terminal field cases of (A) sheep scrapie (65 mg), (B) cattle BSE (30 mg), (C) deer CWD (90 mg) and (D) pre-mortem sheep scrapie (65 mg), together with urinary protein from appropriate healthy controls, were fractionated under reducing SDS-PAGE conditions and subsequently analyzed by Western blot using an anti-human a1-ACT monoclonal (A, B & D) or polyclonal (B) antibody. In each case a duplicate blot incubated with HRP- labeled secondary antibody alone was also prepared and is shown. To verify equivalent protein loading, blots were stripped and re-probed with an anti-human albumin antibody. Recombinant a1-ACT (30 ng) was loaded as positive control. Molecular weight marker positions are indicated. Panel A, lane 8 represents urine from a suspected scrapie case, the precise status of which is unclear (U) at present. Pre-terminal sheep scrapie urine (D) was analyzed from six individual healthy sheep and from one case at 10, 9, 2 & 1 months prior to being sacrificed at end-stage of disease (lanes 8–11 respectively). doi:10.1371/journal.pone.0003870.g006 autopsy from a cohort of Swiss patients who died between 1996 and 2004. Patients with prion disease, Alzheimer’s disease and patients without neurologic disorders were included. Tissues were processed according to established guidelines regarding safety and ethics and periods from death until post-mortem tissue collection were approximately 4–18 hours. All tissues were snap-frozen in liquid nitrogen prior to long-term storage at 280uC. Tissues from the RML murine model of prion pathogenesis were collected at various stages throughout pathogenesis. All murine tissues were immediately snap-frozen in liquid nitrogen prior to storage at 280uC until required. Total RNA was isolated using Trizol (Invitrogen AG, Switzerland) from eight patients of each control, AD and sCJD group. Preparation of labeled cRNA and microarray hybridization All procedures for preparation of labelled cRNA probes and subsequent Genechip hybridisations were performed according to suggested Affymetrix guidelines (http://www.affymetrix.com). To- tal RNA isolated from whole brain using Trizol was subjected to a purification step (RNeasy mini kit, Qiagen, Switzerland). Quality of total RNA was assessed by agarose gel electrophoresis. Equal amounts of total RNA from 3 individual brains of mice at 145 dpi stage of prion pathogenesis were combined to act as template for generation of probe for one biological replicate. Thus, from nine mock-inoculated and nine prion inoculated mice, a total of three sets of labelled cRNA probes were synthesized representing three biological replicates. Double-stranded cDNA was synthesized using 20 mg total RNA as template (cDNA Synthesis kit, Cat.No.11917- 010, Invitrogen, Switzerland) and primer 59-GGCCAGT- GAATTGTAATACGACTCACTATAGGGAGGCGG(dT)24-39. Biotin-labeled cRNA was synthesized, using Enzo BioArray High- Yield RNA transcript labeling Kit (T7) (Enzo Life Sciences, Germany), purified using the RNeasy mini kit and quantified by spectrophotometry. Quality was assessed by agarose gel electro- phoresis. Typical yields were in the range of 120–150 mg labeled cRNA. 15 mg of labeled cRNA was fragmented in 40 mM Tris- acetate, 100 mM KOAc, 30 mM MgOAc, pH 8.1 at 95uC for 35 min. Following fragmentation, an aliquot of one representative of the replicates of each sample was hybridized to Affymetrix Test3 arrays to determine the quality of the probes, reflected by the 39/59 ratios, and to test all buffers using conditions outlined in the Affymetrix expression analysis manual. The samples were then hybridized to Affymetrix MOE430A and MOE430B chips. All hybridizations were carried out for 16 h at 45uC at 60 rpm. Chips were then washed and incubated with streptavidin-phycoerythrin according to the manufacturers instructions on the Affymetrix GeneChip Fluidics Station 450 with and scanned using the Affymetrix GS Scanner 2500 in conjunction with Affymetrix Microarray Suite 5.0 software. PLoS ONE | www.plosone.org 10 December 2008 | Volume 3 | Issue 12 | e3870