In-vivo Gene Signatures of Mycobacterium tuberculosis In C3HeB/FeJ Mice
Ontology highlight
ABSTRACT:
ORGANISM(S): Mycobacterium tuberculosis H37Rv
SUBMITTER: Deepak Kaushal
PROVIDER: E-GEOD-70765 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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A minimum of two independent biological replicates were studied for each surfactant preparation. Purified human SP-A was also examined for its effects on Mtb transcription, and phosphate buffered saline (PBS) was used as a âno treatmentâ control. In experiments with SP-A (in 650 µl PBS), the pure apoprotein was either pre-coated on the flask as done with the surfactant preparations, or was directly added and incubated with Mtb during study. Mtb was added to the flask in 3.35 ml of DMEM (supplemented with 2% FCS and 5 mM HEPES) and exposed to coated surfactant/SP-A or PBS control for 30 min or 2h at 37oC, followed by RNA isolation as detailed by Rohde et al. (2008). GTC lysis/stabilization buffer was added, and pelleted Mtb were washed and then digested with lysozyme. TRIzol (Invitrogen) was added, and organisms were disrupted using glass beads in a Mini-Bead Beater (BioSpec Products, Inc. Bartlesville, OK). After extraction of Mtb lysates with chloroform, ethanol was added to the aqueous phase before loading samples onto RNeasy mini columns (Qiagen, Valencia, CA). Subsequent steps were then performed according to the manufacturerâs instructions. Following elution with RNase-free water, the RNA samples were treated with DNase (Turbo DNAfree, Applied Biosystems, Foster City, CA), and stored at -70oC until analysis. The MessageAmpTMII-Bacteria system (Applied Biosystems) was used to amplify mycobacterial RNA for all samples [16]. Total RNA was reverse transcribed to yield double-stranded amino-allyl modified aRNA, which was labeled with Alexa Fluor 555 and Alexa Fluor 647 (Invitrogen). Excess reactive dyes were removed with a Mega Clear purification kit (Applied Biosystems). Next, Alexa-labeled aRNA from paired samples was dried using a Speedvac, and re-suspended in hybridization buffer (5x SSC, 25% formamide, 0.15% SDS, and 25 μg salmon sperm DNA). The mixed samples were denatured by being heated to 95oC for and then cooled to 50oC prior to loading them onto microarrays. The arrays were hybridized for approximately 15 h at 45oC, washed and then dried by centrifugation. Fluorescence intensity data from each array were collected with a GenePix 4000B scanner (Axon Instruments, Union City, CA) using GenePixPro3.0 software (Axon Instruments). Image analysis, i.e. spot identification, background measurements, quality assessment and flagging were conducted with Imagene software (Version 6.0, BioDiscovery, Inc., El Segundo, CA). Poor quality spots were filtered and removed from the analysis. Subsequent normalization, statistical analysis (i.e. t-test), and visualization of array data was conducted using Genespring (Version 7, Agilent Technologies, Palo Alto, CA).
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