animal: Human tissue: Skin condition: Pus group: C subgroup: E
Treatment protocol
NA
Growth protocol
Cutaneous abscess samples were collected from patients undergoing incision and drainage procedure as part of clinical care at the San Francisco General Hospital, and were frozen in RNA-later (Qiagen, Valencia, CA) until RNA isolation. For RNA isolation from infected mouse kidneys, C57Bl/6 mice (Charles River, Wilmington, MA) were injected intravenously with 100 μL phosphate-buffered saline (PBS) containing 1 × 108 colony forming units (CFUs) USA300 from 4 hours’ cultures in TSB. Three days later, kidneys were frozen in RNA-later until RNA isolation. To start the liquid Staphylococcus aureus USA300 control cultures, single colonies from tryptic soy agar (TSA) plates supplemented with 5% sheep blood were inoculated into tryptic soy broth (TSB) and incubated at 37°C while shaking at 200 rpm for the indicated times. The liquid cultures were grown to mid log phase [4 hours].
Extracted molecule
total RNA
Extraction protocol
Frozen tissues in RNAlater were homogenized using a gentleMACS cell dissociator (Miltenyi), followed by 10 minutes’ incubation at room temperature (RT) in nuclease-free PBS (Ambion, Grand Island, NY) containing 0.05% Triton-X100 (Thermo), 10 U/mL RNAse inhibitor RNAseIn (Promega, Madison, WI) and 20 mg/mL DNAseI grade II (Roche). The suspensions were passed through a 40-μm filter (BD Biosciences, San Jose, CA), washed twice with PBS containing 10 U/mL RNAse inhibitor RNAseIn, incubated for 5 minutes at RT in RNA Protect Bacteria Reagent (Qiagen), pelleted, and frozen. Bacteria were lysed by incubation for 30 minutes at 37°C in 30 mM Tris ( pH 8.0) containing 1 mM EDTA, lysostaphin (Sigma, St. Louis, MO) at 5 mg/mL, and protease K (Qiagen). RNA was isolated using RNAEasy RNA purification kit (Qiagen). Bacterial RNA was further enriched using the MicrobEnrich purification kit (Ambion).
Label
Cy5
Label protocol
Bacterial cRNA was prepared as follows. About 100 ng of bacterial total RNA was polyadenylated and reverse transcribed using MessageAmpTM II-Bacteria kit (Life Technologies, Grand Island, NY). After cDNA synthesis, cRNA labeled with Cy3 or Cy5 was synthesized using T7 RNA polymerase (Agilent). The labeled cRNA was purified on an affinity resin column (Qiagen). Cy-dye incorporation and yield of labeled cRNA were measured with the ND-1000 spectrophotometer.
Channel 2
Source name
Staphylococcus aureus USA300 In Vitro reference pool
reference: Staphylococcus aureus USA300 In Vitro reference pool
Treatment protocol
NA
Growth protocol
To start liquid cultures, single colonies from tryptic soy agar (TSA) plates supplemented with 5% sheep blood were inoculated into tryptic soy broth (TSB) and incubated at 37°C while shaking at 200 rpm for the indicated times.
Extracted molecule
total RNA
Extraction protocol
Frozen tissues in RNAlater were homogenized using a gentleMACS cell dissociator (Miltenyi), followed by 10 minutes’ incubation at room temperature (RT) in nuclease-free PBS (Ambion, Grand Island, NY) containing 0.05% Triton-X100 (Thermo), 10 U/mL RNAse inhibitor RNAseIn (Promega, Madison, WI) and 20 mg/mL DNAseI grade II (Roche). The suspensions were passed through a 40-μm filter (BD Biosciences, San Jose, CA), washed twice with PBS containing 10 U/mL RNAse inhibitor RNAseIn, incubated for 5 minutes at RT in RNA Protect Bacteria Reagent (Qiagen), pelleted, and frozen. Bacteria were lysed by incubation for 30 minutes at 37°C in 30 mM Tris ( pH 8.0) containing 1 mM EDTA, lysostaphin (Sigma, St. Louis, MO) at 5 mg/mL, and protease K (Qiagen). RNA was isolated using RNAEasy RNA purification kit (Qiagen). Bacterial RNA was further enriched using the MicrobEnrich purification kit (Ambion).
Label
Cy3
Label protocol
Bacterial cRNA was prepared as follows. About 100 ng of bacterial total RNA was polyadenylated and reverse transcribed using MessageAmpTM II-Bacteria kit (Life Technologies, Grand Island, NY). After cDNA synthesis, cRNA labeled with Cy3 or Cy5 was synthesized using T7 RNA polymerase (Agilent). The labeled cRNA was purified on an affinity resin column (Qiagen). Cy-dye incorporation and yield of labeled cRNA were measured with the ND-1000 spectrophotometer.
Hybridization protocol
Cy3 labeled S. aureus USA300 reference and Cy5 labeled sample-derived cRNA (825 ng each) were fragmented following instructions in the Agilent’s In situ Hybridizatyion kit-plus. Hybridization and washing were performed using Tecan HS4800 Pro hybridization station (Tecan, Research Triangle Park, NC).
Scan protocol
Arrays were scanned in the Agilent Surescan Microarray Scanner.
Description
Staphylococcus aureus USA300 derived from Human Skin Pus - InVivo
Data processing
Array images were extracted using the Agilent Feature Extraction software version 10.7. Data from infected tissues were first normalized by calculating ratios of fluorescence signal of test RNA versus Reference, and then expressed as linear fold change relative to values from USA300 from 4 hours’ subculture in TSB (n = 3), also normalized to Reference. For studies in infected mouse kidneys, USA300 from 4 hours’ subculture in TSB served as infection inoculum, representing the start of infection. Microarray data were analyzed using the Bioconductor package. The fold change was derived from the fitted coefficient on the treatment term, and significance was assessed via Limma’s moderated F statistic.
