7 The second and larger piece was formalin-fixed, stained with hematoxylin and

eosin (H&E) and trichrome, and graded and staged for fibrosis (Ishak fibrosis and Metavir) by an experienced hepatopathologist. Tissues that were of poor quality or of inadequate length were excluded before review. Metavir scores from subsequent biopsies and intervening Fibroscan values for the same subjects confirmed staging results from the first biopsy. In preparation for LCM, tissues were thawed and sectioned at −24°C. Seven- and 10-μm sections were made onto polyethylene naphthalate membrane slides, fixed in 70% ethanol, and hematoxylin-stained for morphologic and nuclear detail. The duration of room temperature exposure before LCM was ≤10 minutes. The Leica LMD6000 laser capture microdissecting system was used for LCM. Two portal tracts and six parenchymal segments were captured per subject. Parenchymal segments were selleck chemicals llc captured according to anatomic landmarks and hepatic zonation to include two periportal segments, two midzonal segments, and two centrilobular segments. Segments were captured in Trizol buffer and stored at −80°C until RNA isolation. RNA was isolated from each sample separately using the Agencourt RXDX-106 ic50 RNAdvance Cell v. 2 system (Beckman Coulter Genomics, Danvers, MA). Isolated RNA from each sample was divided into three aliquots.

The first was tested for housekeeping transcripts using quantitative polymerase chain reaction (qPCR). The second was tested for HCV RNA using the ABBOT RealTime HCV Assay. The third was linearly amplified using the NuGEN Ovation Pico WTA System (NuGEN Technologies, San Carlos, CA). Attempts to quantify RNA using a NanoDrop (Thermo Scientific NanoDrop Products, Wilmington, DE) were unsuccessful because of the small numbers of captured cells; therefore, qPCR for GAPDH messenger RNA (mRNA) was used to estimate the number of captured cells after standardizing to a known quantity of Hepatoma 3B cells in culture, resulting in the estimate of ≈20 copies per cell. Samples were selleck submitted to the Johns Hopkins Microarray

Core Facility and hybridized to the GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). The mean slope of 5′ intensity versus 3′ intensity was 0.07 ± 0.06, indicating no preferential mRNA degradation. Results of the microarray were validated by performing qPCR using SyBR Green and gene-specific primers on 1/8 hepatic parenchyma sections from each subject. Raw data from microarrays is available on NCBI GEO via accession number GSE33650 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=pnmnhuoacgiaglk&acc = GSE33650). SBA, a surrogate for protein expression, was measured on archived specimens at a dilution of 1:500 using the DetectX Butyrylcholinesterase Fluorescent Activity Kit (Arbor Assays, Ann Arbor, MI).