genotype/variation: WT cell type: common lymphoid progenitors
Treatment protocol
The RNA samples of CLPs cells were pooled from WT or DD mouse (rep1=WT8,DD8,rep2=WT7,DD7 and rep3=WT6,DD6). lineage+ cells (CD4, CD8, CD3e, CD11b, Gr-1, Ter119, B220, NK1.1) were partially removed using sheep anti-rat IgG conjugated magnetic beads (Dynabeads M-450, Invitrogen), and the remaining cells were stained with avidin-PerCP-Cy5.5 (BD). The cells were stained with FITC-conjugated anti Sca-1 (D7; BD), PE-conjugated anti CD135 (A2F10; eBioscience), APC-conjugated anti CD117 (2B8; BD) and PE-Cy7-conjugated anti CD127 (A7R34; eBiosciences or TONBO). Then they were sorted by FACSAria II (BD) into 3% FBS/PBS.
Extracted molecule
total RNA
Extraction protocol
RNA was prepared using the RNeasy Plus Micro kit (QIAGEN) following the manufacturer's recommendations. RNA quantity and quality were measured with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
Three RNA samples of CLPs or MPPs were performed in two rounds of amplification using the Amino Allyl MessageAmpII aRNA Amplification Kit (Ambion) and Cy3 labeled with a CyDye post-labeling Reactive Dye Pack (Amersham) following the manufacturer’s protocol. After amplification and labeling, cRNA yields and dye incorporation efficiencies were determined using a Nanodrop spectrophotometer.
Hybridization protocol
600 ng of Cy3-labelled cRNA (specific activity > 104-156 pmo Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Sureprint G3 Mouse GE 8x60k Microarray slides for 17 hours at 65°C 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 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Labeled samples were incubated with Sureprint G3 Mouse GE 8x60k Microarray Kit slides for 17 h. The slides were scanned, and the gene expression analysis was performed using the Genespring GX software program version 11.
Description
Gene expression in CLPs of WT1
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
