|Application:||Gene expression microarray analysis|
|Number of samples:||90|
|Release date:||Apr 24 2014|
|Last update date:||Aug 16 2018|
|Diseases:||Simian Acquired Immunodeficiency Syndrome|
|Dataset link||Role of TCR:peptide/MHC Class I Affinity in Directing Different Maturation Phenotypes of Dominant and Subdominant Epitope-Specific CD8+ T Cells in Mamu-A*01+ Rhesus Monkeys|
Peripheral blood was collected on days 14, 21, 28, 35, 42, 56, 70, and and 210 after inoculation with SIVmac251. Plasma viral RNA from these samples was measured using an ultra-sensitive branched DNA amplification assay (Siemens Diagnostics, Berkeley, CA). PBMC were stained with p11C and p54AS tetramers and CD3 and CD8 antibodies at 4°C. Tetramer-positive single CD3+CD8+ lymphocytes were sorted to greather than 95% purity into RNAprotect (Qiagen) at 4°C. RNA was isolated using a Trizol (Invitrogen) extraction protocol. Briefly, 0.8 ml of Trizol were added to the cell pellet and incubated for 5 minutes at room temperature and 0.16 ml of chloroform were added, shaken vigorously by hand for 15 seconds, incubated at room temperature for 2-3 minutes and centrifuged at 13,000 rpm for 15 minutes at 4ºC. The colorless upper aqueous phase was carefully collected and transferred to a new tube containing 2 ?l of linear acrylamide for mixing. An equal volume of isopropyl alcohol was then added and mixed. The mixture was incubated at room temperature for 10 minutes and centrifuged at 13,000 rpm for 10 minutes at 4ºC. The supernatant was collected and the RNA was washed with 1 ml of 70% ethanol and centrifuged at 10,500 rpm for 5 minutes at 4ºC. The supernatant was completely removed and the RNA pellet was allowed to air-dry. The RNA was then resuspended in RNase-free water and stored at -80ºC. RNA integrity was tested using an Agilent Bioanalyzer. RNA was then amplified using the TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre Biotechnologies) according the manufacturer’s instructions. Amplified biotinylated antisense-RNA (aRNA) was resuspended in RNase-free water and stored at -80ºC. Nanodrop ND-1000 was used to determine the biotinylatedaRNA concentration and an Agilent Bioanalyzer was used to determine its integrity.Amplified aRNA was hybridized to Illumina Human HT-12 Expression BeadChips according to the manufacturer instructions and was stained with Streptavidin Cy3 for detection (Illumina, San Diego, CA, USA).
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