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Transcriptomic analysis of host response to the Karp strain of Orientia tsutsugamushi in inbred mice blood
Author(s) -
Miller StacyAnn,
Chao ChienChung,
Yang Ruoting,
Chan TeikChye,
Zhang Zhiwen,
Chen HuaWei,
Ching WeiMei,
Hammamieh Rasha,
JettTilton Marti
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1076.4
Subject(s) - biology , orientia tsutsugamushi , gene expression , rna extraction , microarray , microarray analysis techniques , dna microarray , gene , microbiology and biotechnology , transcriptome , real time polymerase chain reaction , gene chip analysis , gene expression profiling , scrub typhus , virology , genetics
Scrub typhus is caused by the obligate intracellular Gram negative bacterium, Orientia tsutsugamushi ( OT ) . Previous studies have shown mRNA expression profile was altered in PBMC upon in vitro infection by OT. A combined expression profile based on a few selected mRNAs appeared unique to OT infection. To better understand the host response upon infection, we infected C3H mice and monitored gene expression profile in blood at different time post infection. C3H mice were infected with sham (i.e. PBS only) or Karp strain of OT (250 mLD 50 ) by an artificial route. Mice were sacrificed at different time post infection. RNA was isolated from blood using the automated PAXgene blood miRNA kit on the Qiacube. Agilent SurePrint G3 Mouse GE 8×60K Microarray slides were used. The experimental and universal reference RNA were labeled with CY5 and CY3, respectively using Agilent Low Input Quick Amp Labeling Kit and Two‐Color Spike‐Mix, following by fragmentation and hybridization using the Agilent Gene Expression Hybridization Kit. Arrays were scanned using the Agilent Microarray G4900DA scanner system. Microarray data was extracted using Feature Extraction v 11.0.1.1. Further analysis was performed using functional analysis clustering toolbox (FACT) and customized R code. Using FACT, we were able to dissect the most important gene expression patterns. Lots of changes (1667 probes) occurred only at 6 h, 12 h (796 probes), then 2 days (828 probes) post infection. Multiple pathways with highly up‐regulated expression occurred at 8 – 12 h. Pathway‐specific qPCR is being performed to confirm these altered mRNAs. Using a mouse model with an artificial route of infection, we are able to show various genes involved in pathways responded to OT infection. While further validation of these altered genes is needed, the results demonstrated a massive change of gene expression early during the infection progression. Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the author(s) and do not constitute endorsement by the U.S. Army.