Computational protocol: Mosquito Cellular Factors and Functions in Mediating the Infectious entry of Chikungunya Virus

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Protocol publication

[…] Upon infection, C6/36 cells were harvested at 0 min, 15 mins, 30 mins and 120 mins post infection (pi). At 0 min pi, cells were harvested immediately upon virus inoculation. At each time point, C6/36 cells were washed with 2 ml of the pre-warmed (28°C) maintenance medium. After decanting the maintenance medium, 1 ml of Qiagen Cell Protect solution was added to each flask. Detached cells were transferred into a sterile 2 ml tube and were stored immediately at −80°C until total RNA extraction. Cells were homogenized in 350 µl RLT buffer in QIAshredder spin columns (Qiagen, Hilden, Germany) prior to total RNA extraction with Qiagen RNeasy Protect cell mini kit (Qiagen) according to manufacturer's instructions. Hundred nanograms of total RNA were used for probe synthesis of cy3-labeled cRNA, and hybridizations were carried out on an Aedes mosquito customized gene expression microarray (18760 transcripts from Vector Base Aedes aegypti database with 2 best probes per transcript) in Agilent GE 8×60K array format (Agilent Technologies, California, USA). Hybridization was carried out at 65°C for 17 hours in an Agilent hybridization oven at 10 rpm. After hybridization, microarrays chips were washed in gene expression wash buffer 1 for 1 min at room temperature and 1 min in gene expression wash buffer 2 at 37°C before scanning on the Agilent High Resolution Microarray Scanner (C-model). Raw signal data was extracted from the TIFF image with Agilent Feature Extraction Software (V10.7.1.1). The raw microarray data was processed and analyzed with Partek Genomics Suite (Partek, St Louis, Missouri, USA) to generate values representing fold changes in gene expression. An average of the duplicate values was used to calculate fold change, and each value was then assessed for its statistical significance, using analysis of variance (ANOVA). Host genes demonstrating at least a 1.5-fold change in expression upon CHIKV infection were selected for further investigation. Pathway analysis was subsequently detailed with Ingenuity Pathway Analysis (IPA) 9.0 (Ingenuity Systems 2011, Redwood City, California) and differentially regulated genes involved in the clathrin-mediated endocytic pathway were selected for pathway mapping. […]

Pipeline specifications

Software tools Agilent Feature Extraction, Partek Genomics Suite, IPA
Application Gene expression microarray analysis
Organisms Chikungunya virus
Diseases Chikungunya Fever
Chemicals Chlorpromazine