burnetii NMII proteins. The 48-72 hpi time frame was used because (i) C. burnetii would be in logarithmic growth [6] and (ii) (ii) previous studies have shown observable changes in PV size within C. burnetii infected Vero cells when treated overnight with 10 μg/ml of CAM at 48 hpi [7]. RNA extraction, microarray hybridization and data analysis Following the infection and treatment protocols (Figure 1), total RNA was isolated using Tri-Reagent (Ambion, Austin,
TX) according to the manufacturer’s recommendations. All RNA samples were DNase treated using RQ1 DNase (Promega, Madison, WI) https://www.selleckchem.com/products/ag-881.html and confirmed DNA free by PCR. RNA integrity was assessed by electropherogram using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California). Total RNA (500 ng) from each sample was then amplified using an Epicentre® Biotechnologies (Madison,
WI) TargetAmp™ 1-Round AminoallylaRNA PRIMA-1MET supplier Amplification Kit, yielding approximately 6-10 μg of aminoallyl-aRNA (AA-aRNA). Alexa Fluor® 555-GREEN (Invitrogen, Carslbad, CA) was used to label the uninfected AA-aRNA, while Alexa Fluor® 647-RED (Invitrogen) was used to label the AA-aRNA from the C. burnetii infected cells. Labeled AA-aRNA (2 μg) with a dye incorporation efficiency range of 18-34 picomol/microgram, were mixed pair-wise and hybridized overnight to Human OneArray™ microarrays (Phalanx Biotech Group, Palo Alto, CA). Human OneArrays contain 32,050 oligonucleotides; 30968 human genome Baf-A1 mouse probes and 1082 experimental control probes formed as 60-mer sense-strand DNA elements. Arrays were hybridized, washed, and dried rapidly according to the manufacturer’s instructions. Six hybridizations for each condition set (CAM and mock treated) were performed with three biological and two technical replicates. Signal VX-661 chemical structure intensity of the hybridized arrays were measured by ScanArray Express (PerkinElmer, Boston, MA, USA) and the images were processed using GenePix Pro version 4.0 (Axon, Union City, CA, USA). The processed GenePix Pro 4.0 output was further analyzed using Loess-Global intensity dependent normalization through the GenePix Auto Processor (http://darwin.biochem.okstate.edu/gpap3/)
as previously described [25–27]. Normalized ratio values for each data point were averaged across the three biological replicates and two technical replicates. Significant expression differences were defined as a P-value < 0.05 and displayed as a fold change of ≥2 fold [28, 29]. The microarray data were deposited at the NCBI Gene Expression Omnibus (GEO) under the platform accession number GPL6254 and the series number GSE23665. The biological function of the identified genes was determined bioinformatically by the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov/) [30] as well as by Ingenuity pathway analysis (Ingenuity® Systems, http://www.ingenuity.com).