Computational protocol: Genomic Analysis Reveals a Potential Role for Cell Cycle Perturbation in HCV-Mediated Apoptosis of Cultured Hepatocytes

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Protocol publication

[…] Microarray format, protocols for probe labeling, and array hybridization are described at http://expression.microslu.washington.edu. Briefly, a single experiment comparing two mRNA samples was done with four replicate Human 1A (V2) 22K oligonucleotide expression arrays (Agilent Technologies) using the dye label reverse technique. This allows for the calculation of mean ratios between expression levels of each gene in the analyzed sample pair, standard deviation and P values for each experiment. Spot quantitation, normalization and application of a platform-specific error model was performed using Agilent's Feature Extractor software and all data was then entered into a custom-designed database, Expression Array Manager, and then uploaded into Rosetta Resolver System 7.0 (Rosetta Biosoftware, Kirkland, WA) and Spotfire Decision Suite 8.1 (Spotfire, Somerville, MA). Data normalization and the Resolver Error Model are described on the website http://expression.viromics.washington.edu. This website is also used to publish all primary data in accordance with the proposed MIAME standards . Selection of genes for data analysis was based on a greater than 95% probability of being differentially expressed (P≤0.05) and a fold change of 2 or greater. The resultant false positive discovery rate was estimated to be less than 0.1% (Walters, unpublished data). Ingenuity Pathway Analysis (IPA) software and Entrez Gene (www.ncbi.nlm.nih.gov/sites) were used for gene ontology analysis. [...] Quantitative real-time PCR (RT-PCR) was used to validate the gene expression changes and measure intrahepatic HCV RNA. Total RNA samples were treated with DNA-free DNase Treatment and Removal Reagents (Ambion, Austin, TX). Reverse transcription was performed using random hexamer primers and Taqman RT reagents (Applied Biosystems, Foster City, CA). Real-time PCR was performed using an ABI 7500 Real Time PCR system and Taqman chemistry. Each target was run in quadruplicate with Taqman 2× PCR Universal Master Mix and a 20 µL total reaction volume. Primer and probe sets for relative quantification were selected from the Assays-on-Demand product list (Applied Biosystems) including two endogenous controls, GAPDH and 18 S ribosomal RNA. Quantification of each gene, relative to the calibrator, was calculated by the instrument, using the equation 2−ΔΔCT within the Applied Biosystems Sequence Detections Software version 1.3. Probes used for analysis (Applied Biosystems): Human genes: eukaryotic 18S rRNA (Catalogue No. Hs99999901_s1); ATF3 (catalogue No. Hs00910173_ml), MKi67 (catalogue No. Hs01032443_ml), MCM4 (catalogue No. Hs00381539_ml), MCM6 (catalogue No. Hs00195504_ml), TGIF1 (catalogue No. Hs00545014_ml), CAT (catalogue No. Hs00156308_ml), SMAD7 (catalogue No. Hs00998193_ml), GADD45A (catalogue No. Hs00169255_ml), GADD45B (catalogue No. Hs00169587_ml)Primer and probe sets for absolute quantification of intrahepatic viral load were designed based on sequences of HCV 1a armored RNA (Ambion Diagnostics, Austin, TX) using Primer Express (version 3). A standard curve was made from six serial dilutions of HCV 1a armored RNA (Ambion Diagnostics) with a known viral copy number. The PCR efficiency was determined by the slope of the standard curve Standard curve analysis and viral load was determined using the Applied Biosystems SDS Software 1.3 (Applied Biosystems, CA). Total RNA was DNase treated prior to cDNA synthesis via reverse transcription and all samples were processed with equal mass amounts of total RNA . All measurements were taken in quadruplicate with negative and non-template controls. Primer and probe sets consisted of F: CAC TCC CCT GTG AGG AAC TAC TG, R: GCT GCA CGA CAC TCA TAC TAA CG, and P: 6FAM-TTC ACG CAG AAA GC-MGBNFQ and were designed from the 5′UTR using Primer Express 3.0 (Applied Biosystems, CA). Quantification of HCV RNA levels was performed on the same total RNA sample that was used for the microarray experiments. […]

Pipeline specifications

Software tools IPA, Primer Express
Applications Gene expression microarray analysis, qPCR
Organisms Classical swine fever virus, Homo sapiens
Diseases Infection, Liver Diseases, Chemical and Drug Induced Liver Injury