monkey: cynomolgus subject id: 9F08 gender: Male infection type: Mock time point (day): 2
Treatment protocol
Infection of cynomolgus macaques and sample preparation has been described extensively (11, 12). Briefly, animals were GAS-culture negative and had negligible antistreptolysin O titers, indicating no recent history of GAS exposure. Twenty animals were subjected to a mock-inoculation protocol (PBS only) for 5 weeks, rested for 4weeks, and inoculated in the upper respiratory tractwith 107 CFUsMGAS5005. Blood, saliva, and throat swabs were collected on days 0, 1, 2, 4, 7, 9, 16, 23, 32, 45, 58, 72, and 86.Only the first nine time-points were studied because specimens collected during days 0 to 32 had matching comparator specimens from the mock-infection protocol. Thirty-two clinical and laboratory parameters were measured by the same veterinarian during mock and infection periods
Growth protocol
GAS strain MGAS5005 (GenBank CP000017), genetically representative of contemporary serotype M1 strains, was grown as previously described (11, 12)
Extracted molecule
total RNA
Extraction protocol
Tonsil swabs were immediately frozen on dry ice.The swabs contained a mixture of epithelial and inflammatory cells, but microscopic examination indicated that the majority of cells were epithelial cells. Host cells were lysed using the FastPrep FP 120 system and Lysing Matrix D Tube (MP Biomedicals) with 500 μL phenol:chloroform (5:1) at pH 4.5 (Applied Biosystems), 500 μL CRSR-”GREEN” (MP Biomedicals), and 300 μL of 5 mM arunocarboxylic acid. RNA was purified in 96-well format as described (1). RNA quantity was determined with an Agilent 2100 Bioanalyzer System (Agilent Technologies)
Label
biotin
Label protocol
All tonsil swab RNA (average 4.2 μg per swab) was used for cDNA synthesis as described (2). In vitro transcription labeling with biotinylated UTP and CTP was performed according to the manufacturer’s recommendations (Enzo Diagnostics) for 10 h at 37 °C. Amplified cRNA was purified as described above. The quality and size of cRNA was verified with an Agilent 2100 Bioanalyzer System (Agilent Technologies)
Hybridization protocol
hybridized at a constant temperature of 45◦C for approximately 16 hours
Scan protocol
GeneChips were scanned using the standard Affymetrix GeneChip Scanner
Description
Gene expression data from_sample from sample from subject 9F08, sex= Male, infection= Mock, day= 2
