Dataset features


Application: RNA-seq analysis
Number of samples: 3
Release date: Dec 5 2010
Last update date: May 23 2013
Access: Public
Diseases: Respiratory Tract Infections, Porcine Reproductive and Respiratory Syndrome
Dataset link Aberrant host immune response induced by highly virulent PRRSV identified by digital gene expression tag profiling

Experimental Protocol

Nine conventionally-reared, healthy 6-week-old, crossbred weaned pigs (Landrace×Yorkshire) were selected from a high-health commercial farm. All pigs were PRRSV-seronegative determined by ELISA (HerdChek PRRS 2XR; IDEXX Laboratories) and absence of PRRSV tested by RT-PCR. Pigs were randomly assigned to three groups in the experiment and raised in isolation rooms. Six pigs were inoculated with 6 ml viral suspension (4 ml intranasally and 2 ml intramuscularly) of H-PRRSV at a dose of 106.0 TCID50 ml-1 on day 0. Three negative control pigs were treated similarly with an identical volume of DMEM culture media from uninfected MARC-145 cells 1 day prior to experimental infection, and were immediately necropsied.H-PRRSV-inoculated pigs were clinically examined daily and rectal body temperatures were recorded from days -2 to 7 post infection (pi).Three infected pigs randomly chosen within each group were necropsied at each time point of 96 h pi and 168 h pi. Lung samples were collected from uninfected negative control group (C), three pigs at 96 h pi (H96), three pigs at 168 h pi (H168) and immediately frozen in liquid nitrogen for RNA isolation or fixed in 10% neutralized buffered formalin for histological processing. Total RNA was extracted from frozen lungs using standard protocols (Trizol) and then treated with DNase to remove potential genomic DNA contamination according to the manufactures’s protocols. RNA integrity and concentration were evaluated by Agolent 2100 Bioanalyzer.For RNA library construction and deep sequencing, RNA samples were prepared as follows: for each time point of H-PRRSV-inoculated group and UNC group equal quantities of RNA isolated from three individual lungs were pooled. Approximately 6 μg of RNA representing each group were submitted to Solexa (now Illumina Inc.) for sequencing.










Yaosheng Chen

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