Computational protocol: Japanese encephalitis virus infection induces changes of mRNA profile of mouse spleen and brain

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Protocol publication

[…] The total RNA was isolated from mouse spleens and brains respectively with trizol reagent (Invitrogen) for mRNA Microarray. mRNA hybridization was performed by shanghaiBio Corporation (shanghai, China) with the use of 4 × 44 K Agilent Whole Mouse Genome Oligo Microarray (total 41,174 oligo probes from 41,174 mouse genes). For each sample pair, the experiments were done with two independent hybridizations (Cy3 and Cy5 interchanging labeling). Hybridized arrays were scanned at 5 μm resolution on an Agilent DNA Microarray Scanner (Model G2565BA). Data extraction from images was done by using Agilent Feature Extraction software. Hierarchical cluster, gene ontology and pathway analysis were analyzed by using SAS (ShanghaiBio Analysis System). [...] For selected mRNA RT-qPCR, total RNA from the same samples used in microarray analysis was tested by using ABI 7500 FAST real-time PCR system. PCR primers were designed with Primer Express 2.0 software (Invitrogen). Results are shown as fold change. For mRNA RT-qPCR, experiments were carried out with the PrimerScript RT reagent Kit (TaKaRa) and SYBR Green Realtim PCR Master Mix (TaKaRa) according to manufacture's instruction. The housekeeping gene GHDAP was used for standardization of the initial RNA content of a sample. Relative changes of gene expression were calculated by the following formula, and the data are represented as fold upregulation/downregulation. fold change = 2-ΔΔCt, whereΔΔCt =(Ct of gene of interest, treated -Ct of HK gene, treated)-(Ct of gene of interest, control-Ct of HK gene, control), Ct is the threshold cycle number and HK is the house keeping gene. […]

Pipeline specifications

Software tools Agilent Feature Extraction, Primer Express
Applications Gene expression microarray analysis, qPCR
Organisms Mus musculus