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Expression of inflammatory host genes in Chlamydia trachomatis infected human monocytes

ABSTRACT: Messenger RNA of C trachomatis infected monocytes of the three healthy donors was compared with the corresponding mock-infected samples. For mock infection, SPG buffer was used instead of the C trachomatis suspension, all other procedures were performed identically. Cells were resuspended in solution D and total RNA was extracted by application of phenol:chloroform 5:1 (pH 4.5, Ambion, Austin, TX) followed by precipitation in isopropanol at -80°C for 1 hr and incubation with RQ1 DNase (Promega, Madison, WI) at 37 °C for 1 hr. The signal intensity of the G3PDH housekeeping gene on the microarray membrane was used as indirect quality marker for the RNA utilized as only experiments were included in the analysis that revealed a G3PDH signal intensity within 1.5 times the standard deviation of all membranes evaluated in our study. The entire microarray procedure and its analysis has recently been validated and reported in detail. Total RNA of all donors was pooled and for each microarray experiment 150 µg were reverse transcribed and amplified by SMART™-PCR (BD Biosciences Clontech, Palo Alto, CA), a technology that allows reverse transcription of small amounts of total RNA and subsequent amplification of the entire cDNA. Probes were labeled with 32P and subsequently over night hybridized to a filter-based nylon membrane containing immobilized cDNA-specific sequences from a total of 1,184 genes (Human Atlas Array 1.2, BD Biosciences Clontech, Palo Alto, CA). For analysis, we focused solely on the 159 cytokines, chemokines and their receptors as given by the manufacturer. Signal intensities were retrieved by a STORM 860 scanner (Molecular Dynamics, Sunnyvale, CA) in combination with the AtlasImage 2.0 software (BD Biosciences Clontech). Data from all signal intensities were then subtracted by the local background intensity measured around each gene. The local background intensities of all individual genes were subsequently averaged, resulting in the mean background intensity of a particular membrane. Gene expression in our experiments was determined by a spot intensity of a single transcript that exceeded twice the mean background. Normalisation to background and to the G3PDH housekeeping gene was achieved by the global (sum) normalization method. Signal intensity data are given as the ratio of C trachomatis infected vs mock infected probes. Keywords: expression analysis

ORGANISM(S): Homo sapiens

PROVIDER: GSE7601 | GEO | 2007/04/26

SECONDARY ACCESSION(S): PRJNA100167

REPOSITORIES: GEO

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Expression of inflammatory host genes in Chlamydia trachomatis infected human monocytes

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