DNA Microarray Project: Hybridization Reproducibility

Overview

The component 4 of BayGenomics has developed protocols to prepare printed oligonucleotide microarrays. This set of arrays was run to confirm the reproducibility of the systems developed. All protocols are available on this website.

Large-scale cDNA preps were prepared from normal adult mouse brain and liver total RNA, split over 8 replicate microarrays and processed in parallel. 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 upregulated at least 2-fold in brain: Brain2foldup.xls
3. Genes that are upregulated at least 2-fold in liver: Liver2foldup.xls
4. GenMAPP gene expression dataset available from: www.genmapp.org

Brief description of experimental design

Samples:

Normal adult mouse brain and normal adult liver tissues were isolated from c57/Black6 mice.

RNA prep and cDNA synthesis:

Total RNA was extracted from tissues with Trizol using 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. Liver cDNA was labeled with Cy5 and brain cDNA was labeled with Cy3.

Hybridization:

Performed according to standard operating procedure.

Scanning of Arrays:

All arrays were scanned on an Axon 4000B scanner with both channels set at PMT setting of 500. No normalization was performed. For each channel, the local background was subtracted from the signal and the remainder adjusted to 1 if less than 1.