Comment[ArrayExpressAccession] E-GEOD-28484 MAGE-TAB Version 1.1 Public Release Date 2015-01-05 Investigation Title Microarray analysis of chimeric CD4+CD28null and CD4+CD28+ T-cells in humanized mice. Comment[Submitted Name] Microarray analysis of chimeric CD4+CD28null and CD4+CD28+ T-cells in humanized mice. Experiment Description The major goal of this experiment was to identify the overall changes in gene expression of human CD4+CD28 null T-cells that develop after repetitive [xeno] antigen stimulation, in comparison to normal CD4+CD28+ T-cells. These cells were derived by adoptive transfer of human CD4 T-cells from a normal donor (all CD28+) into immunodeficient mice. Chimeric human CD4+CD28+ were isolated from 4 mice, and chimeric human CD4+CD28 null cells from two of these animals after expansion and T-cell differentiation in vivo. Animals were euthanized, and single cell suspensions of splenocytes derived. CD4 T-cell subpopulations were isolated by fluoresence activated cell sorting, RNA was extrated, labeled and hybridized to whole human gene expression arrays. Overall changes in the gene expression were identified using GEDI (gene expression dynamic inspector). 603 genes were found to be statistically significant (P<0.05) between the CD4+CD28+ and CD4+CD28null cells. Candidate genes were validated using qRT-PCR Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name HERAZO MAYA Herazo-Maya Duncan Xue Person First Name JOSE Jose Steve Jianmin Person Mid Initials DAVID Person Email geo@ncbi.nlm.nih.gov Person Affiliation Yale University Person Address Medicine, Yale University, 300 Cedar Street, New Haven, CT, USA Person Roles submitter Protocol Name P-GSE28484-1 P-GSE28484-4 P-GSE28484-5 P-GSE28484-2 P-GSE28484-3 P-GSE28484-6 Protocol Description The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (GE1_107_Sep09 and Grid: 014850_D_20070820) to obtain background subtracted and spatially detrended Processed Signal intensities. Gene expression data was normalized to the geometric mean of all the samples using cyclic loess, for the normalization and statistical analysis we used the average gene expression values of the replicated probes, the number of probes after the average and normalization process was 29807. ID_REF = VALUE = Normalized signal intensity Labeling reactions were performed for each sample using Agilent Low RNA Input Linear Amplification Kit PLUS, One-Color (5184-3523, Agilent Technologies, Santa Clara, CA). Briefly, with a starting concentration of 200 nanograms of total RNA, an initial cDNA strand was synthesized using a oligo(dT)24 primer containing a T7 RNA polymerase, this cDNA was then used as a template to generate Cy3 labeled cRNA by a reverse transcriptase enzyme. After the cRNA was obtained a purification step was performed using RNeasy Mini Kit (74104, Qiagen, Valencia, CA) followed by measurement of the yield and specific activity of each sample 1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60M-0C for 30 minutes in a reaction volume of 55 ml containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ml of 2x GEx Agilent hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65M-CM-^BM-0C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37M-CM-^BM-0C GE Wash buffer 2 (Agilent), then dried immediately. Blood was drawn by venipuncture in each mice and CD4+CD28 cells were isolated by fluoresence activated cell sorting. After this step 1 ml of TRIzol (15596019, Invitrogen, Carlsbad, CA) was added to each sample and they were snap frozen in -80 celsius. Total RNA was extracted using the TRIzol reagent protocol and purified using the miRNeasy Mini Kit (217004, Qiagen, Valencia, CA) under fully automated sample preparation by the assistance of the QIAcube device (9001292, Qiagen, Valencia CA) following the manufacturerM-bM-^@M-^Ys protocol.After extraction, total RNA yield and quality were evaluated using NanoDrop at 260 nm and the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) Slides were scanned immediately after washing on the Agilent DNA High Resolution Microarray Scanner (G2505C US45102918) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5 M-NM-