test: Enterococcus faecalis OG1RF, aspirate from subdermal chamber 2 hours post-infection time point: 2
Growth protocol
A single colony of E. faecalis OG1RF was inoculated into 10-25 ml BH medium and incubated for approximately 15 hours, at which time a volume of the culture was diluted 1:5 into 25-50 ml of fresh BH medium and incubated for 2 more hours. Bacteria were harvested by centrifugation for 15-20 min at 6000 rpm in a Beckman JA-17 rotor at 4°C. Pelleted cells were resuspended to an optical density at 600 nm of ~1.3-1.6 in KPBS and 2 ml were used for each subdermal chamber infection. 2 ml volumes were removed from the subdermal chambers at 2, 4, 8, 24, and 96 hours post-inoculation. ~0.1-0.2 ml aliquots of the initial inoculum and each aspirate were used for serial dilution and plating to quantitate the bacterial load at each time point. Two ml of the initial inoculum, and the remainder of each aspirate (~1.8 ml), were immediately added to 4 ml of RNAprotect Bacteria Reagent (Qiagen Inc., Valencia, CA), vortexed, processed according to the manufacturer’s instructions, flash-frozen in a dry ice/ethanol bath, and stored at -80°C until RNA extraction.
Extracted molecule
total RNA
Extraction protocol
RNA was prepared from subdermal aspirates as follows: frozen pellets were thawed, resuspended in 0.4 ml of RNase-free TE containing 50 mg/ml lysozyme and 1000 U/ml mutanolysin, homogenized using a hand-held motorized homogenizer with RNase-free pellet pestles (Fisher Scientific, Pittsburgh, PA), and incubated for 10 min at 37°C. Each sample was then split in half and extracted in duplicate with the RNeasy Mini Kit (Qiagen Inc.) according to the manufacturer’s instructions with an added homogenization step using the QIAshredder (Qiagen Inc.) immediately after the addition of Buffer RLT. RNA was eluted from each column with two 30 μl volumes of RNase-free water, and total RNA from duplicate extractions of each sample were pooled together. RNA from uninfected serous fluid was extracted with TRIzol Reagent (Invitrogen Corp., Carlsbad, CA) as suggested by the manufacturer immediately following aspiration from the subdermal chamber.
Contaminating DNA was removed using a TURBO DNA-free kit (Ambion, Austin, TX) following the rigorous protocol as directed by the manufacturer. RNA for microarray experiments was then ethanol precipitated in the presence of a glycogen carrier (Roche Applied Science, Indianapolis, IN) and resuspended in 10 μl RNase-free water; 1 μl was reserved for qPCR experiments.
Label
Alexa Fluor 647
Label protocol
Total RNA (9 μl) was reverse transcribed and labeled with Alexa Fluor 647-aha-dUTP using the SuperScript Direct cDNA Labeling System (Invitrogen Corp.). Labeled nucleic acids were purified with the QIAquick PCR purification kit (Qiagen Inc.) and eluted with two 30 μl volumes of 10 mM Tris-Cl, pH 8.5, that were pooled together.
reference: Enterococcus faecalis OG1RF genomic DNA (gDNA)
Growth protocol
gDNA was from a 50 ml culture of E. faecalis grown overnight in beef heart infusion at 37 degrees C.
Extracted molecule
genomic DNA
Extraction protocol
gDNA from a 50 ml culture of E. faecalis grown overnight was extracted using a Genomic-tip 500/G column (Qiagen, Inc.) as directed by the manufacturer. Precipitated DNA was resuspended in 1.7 ml of 10 mM Tris-Cl, pH 8.5. A 1.0 ml volume of gDNA was sheared by nebulization with nitrogen gas, essentially as previously described [9,10], at 25 psi for 4.5 min to obtain fragments of 0.8-1.5 kb. Sheared DNA was ethanol precipitated, resuspended in 0.2 ml sterile water, and stored at -20°C until use in microarray experiments. [9] Mehra S, Lian W, Jayapal KP, Charaniya SP, Sherman DH, et al. (2006) A framework to analyze multiple time series data: a case study with Streptomyces coelicolor. J Ind Microbiol Biotechnol 33: 159-172. [10] Roe B, Crabtree J, Khan A (1996) DNA Isolation and Sequencing: Essential Techniques Series. New York: J. Wiley and Sons.
Label
Cy3
Label protocol
Sheared gDNA, which was used as a reference in the microarray experiments, was labeled with Cy3-dUTP (GE Healthcare Life Sciences, Piscataway, NJ) using the BioPrime Array CGH Genomic Labeling Module kit (Invitrogen Corp.). Labeled nucleic acids were purified with the QIAquick PCR purification kit (Qiagen Inc.) and eluted with two 30 μl volumes of 10 mM Tris-Cl, pH 8.5, that were pooled together.
Hybridization protocol
Labeled cDNA was mixed with 0.5 μg labeled gDNA, dried down to ~10 μl in a speedvac, and mixed with ~40 μl of hybridization probe solution that contained a final concentration of 50% formamide, 5X SSC, 0.1 mg/ml salmon sperm DNA, and 0.1% SDS. The probe was incubated at 95°C for 5 min and cooled at room temperature. Hybridizations were performed at 42°C for 17-19 hours in a MAUI hybridization system (BioMicro Systems, Salt Lake City, UT) using the appropriate MAUI mixers. Slides were then washed twice with 2X SSC, 0.5% SDS, once with 1X SSC, and once with 0.1X SSC before being dried by low-speed centrifugation for 10 min.
Scan protocol
Slides were scanned on a ScanArray 5000 scanner (Perkin Elmer, Waltham, MA).
Description
None
Data processing
Scanned images were imported into BlueFuse version 3.6 or earlier (BlueGnome Ltd., Cambridge, UK) where spots were picked using Bayesian statistical modeling to extract the fluorescence intensities. Data were then filtered based upon intensity, Bluefuse confidence, and spot size. Due to a malfunction during slide printing, block 1 spots were unusable and were removed from the analysis at this step. Replicate spots were fused by averaging the intensities of high quality spots. The filtered raw values were exported for further analysis in Expressionist Analyst version 5.3.2 or earlier software (Genedata Inc., Basel, Switzerland). The ratio of the cDNA intensity to the gDNA intensity for each fused spot was calculated and the data from all chips were quantile normalized, as previously described for analyzing gene expression in bacterial systems [9]. The fold change for each gene was calculated by dividing the cDNA/gDNA ratio obtained at either of the sampled time points (i.e., 2 or 8 hours post-inoculation) by the corresponding cDNA/gDNA ratio of the input inoculum (i.e., 0 hours) for each biological replicate. Individual fold changes were then averaged to obtain the final average fold change. Paired t-tests comparing the cDNA/gDNA ratio for each gene in each time point analysis (i.e., 0 versus 2 hours, 0 versus 8 hours) were performed. Results were filtered for genes showing at least a 2-fold change in expression with a p-value of less than 0.05. For simplicity, data from only those spots known to represent genes in the genome of E. faecalis OG1RF were included in the final analysis. [9] Mehra S, Lian W, Jayapal KP, Charaniya SP, Sherman DH, et al. (2006) A framework to analyze multiple time series data: a case study with Streptomyces coelicolor. J Ind Microbiol Biotechnol 33: 159-172.
