Prion Disease Biomarker Figure 1. Serpin-a3n/a1-ACT transcripts in brain during prion pathogenesis. (A) Northern hybridization analysis of Serpin-a3n RNA expression levels in whole brains of mice throughout pathogenesis following intraperitoneal inoculation with either mock inoculum or inoculum containing 6 log LD50 RML5 mouse prions (dpi; days post-inoculation). For comparison, expression levels of Glial fibrillary acidic protein (Gfap) RNA are also shown. Expression levels were normalized to 18S rRNA levels. Differential expression of Serpin-a3n transcripts is evident between 120 dpi and 130 dpi in whole brain. For comparison, increased RNA levels of Gfap, traditionally thought of as one of the largest gene expression changes during prion pathogenesis, is evident between 120 dpi and 130 dpi and rises to approximately 8-fold by 190 dpi. (B) DNAse-treated total RNA was prepared from whole brains of mock- or prion-inoculated mice at 45, 100, 145 or 190 dpi and used for qPCR analyses. Serpin-a3n RNA levels are progressively elevated in brains of prion infected mice throughout pathogenesis and at 145dpi and 190 dpi are approximately 9-fold and 17-fold higher compared to mock inoculated controls. Murine measurements encompass a total of three biological replicates. All transcript levels were normalized to beta- actin and expressed relative to mock-inoculated mice or non-neurodegenerative controls. In the murine model of prion disease utilized, terminal signs are evident at approximately 200–210 dpi. Error bars represent standard deviations of three biological replicates. (C) Northern hybridization analysis of Serpin-a3n and Gfap in CNS areas of prion infected and uninfected mice at 90 dpi, 100 dpi and 140 dpi stages of pathogenesis. Equivalent loading of RNA is indicated by 18S rRNA levels. Abbreviations: Cb, cerebellum; Sc, spinal cord; Ob, olfactory bulb; Cx, cortex. Cystatin F differential expression in prion infected mice is first evident in the spinal cord between 90 dpi and 100 dpi (8-fold increase). By 140 dpi, Sepina3n RNA levels in spinal cord, cerebellum and cortex are approximately 6-fold, 3-fold and 5-fold respectively. For comparison, by 140 dpi, Gfap RNA levels, an indicator of gliosis, in spinal cord, cerebellum and cortex are approximately 3-fold, 4-fold and 4-fold respectively. No change in expression levels of these transcripts in olfactory bulb at any of the stages of pathogenesis analyzed here. (D) DNAse-treated total RNA was prepared from occipital cortex obtained at autopsy from patients suffering from non-neurodegenerative diseases (Control), Alzheimer’s disease (AD), or sporadic CJD (sCJD) patients. Left panel: Age distribution of the patients in each group at time of death. Right panel: box and whisker plots indicating upper- and lowermost values with mean and 75th percentile for eight patients within each group. Values were tested for statistical significance using Student’s t test (two samples, equal variance). Triplicate qPCR reactions were performed for each sample. doi:10.1371/journal.pone.0003870.g001 PLoS ONE | www.plosone.org 3 December 2008 | Volume 3 | Issue 12 | e3870