DNA Microarray Project: Effect of Cell Sorting

Overview

A number of experiments are planned that require performing cell sorting before analysis of samples on microarrays. This experiment was performed to determine the effect of handling in a cell sorter on murine DO 11.1 hybridoma cells. The following summary Excel® tables of selected data are available:

1. List of genes on the arrays: Master_6868_v1.0.xls
2. Genes that are up or down regulated at least 2-fold in response to cell sorting: Sorting2foldChanges.xls
3. GenMAPP gene expression dataset available from: www.genmapp.org

Brief description of experimental design

Samples:

DO 11.1 T cells were washed with PBS and then processed through an ungated Vantage FACS cell sorter in order to collect all cells.

RNA prep and cDNA synthesis:

Total RNA was extracted from treated and untreated cells with Guanidine thiocyanate according to standard procedures. 20 ug of each total RNA was reverse transcribed into cDNA in the presence of amino-allyl dUTP and coupled with Cy3/Cy5 using standard probe synthesis and fluorescent labeling techniques. cDNAs were prepared as dye swaps for each experiment.

Hybridization:

Performed according to standard operating procedure.

Scanning of Arrays:

All arrays were scanned on an Axon 4000B scanner with PMT settings of 570 volts in the Cy3 channel and 620 volts in the Cy 5 channel. The data were filtered in order to exclude features that did not have a signal strength of at least three times the local background in at least one channel. The ratios of treated/untreated median pixel fluorescence intensity were then calculated for each feature found to have sufficient signal strength. These ratios were then normalized by median centering.