Laser Capture Microdissection (LCM) was performed from frozen tissue sections with the SL Microtest device using µCUT software MMI). Approximately 10,000 cells were captured for each sample. Serial sections were used if cells could not be obtained from a single section. Total RNA was isolated from captured cells with the RNAqueous Micro kit (Ambion) and treated with DNAse I according to the manufacturer's instructions. RNA quantification was perfomed using Ribogreen (Molecular Probes).
Label
Cy5
Label protocol
Total RNA (~10ng) in a volume of 25 µl was converted into an OmniPlex WTA cDNA library and amplified by WTA PCR using reagents and protocols according to the beta-commercial TransPlex WTA kit (Rubicon Genomics, Ann Arbor, MI). For each sample, a single 10 µl aliquot of the WTA cDNA library was amplified by WTA PCR and products were purified. Four or five 5 ng aliquots of product were subjected to a second WTA PCR amplification in the presence of amino-allyl dUTP for post amplification labeling and products were pooled before proceeding with the hybridization. Yields after all WTA PCR amplifications were between 2 to 5 ?g per reaction. For all WTA amplified cDNA samples with amino-allyl dUTP incorporated (10-20 ?g), nuclease free water was added to 500 ?l, the sample was transferred to a Microcon YM-30 filter, and centrifuged at 13,000 rcf at RT for 12 minutes. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The amount of sample was measured and the volume was adjusted to 5 ?l with nuclease free water. For labeling, 5 ?l of 1 M sodium bicarbonate (pH 9.0) was added and allowed to incubate at RT for 15 min. Nine ?l of anhydrous DMSO were added to Mono Reactive Cy3 and Cy5 dye packs (Amersham, Buckinghamshire, England), mixed thoroughly, and 1 ?l of the proper dye was added to each sample. The labeling mixture was incubated in the dark at RT for 1 hour. The reaction was stopped by the addition of 9 ?l of 4 M Hydroxylamine for 15 min. For each labeling mixture, RNase free water was added to 100 ?l, 500 ?l of PB buffer was added and Cy3 and Cy5 mixtures were added to separate Qiaquick (Qiagen, Valencia, CA) spin columns and centrifuged at 13,000 rcf. Columns were washed twice with 700 ?l of buffer PE, and centrifuged dry at 13,000 rcf for 2 min. To elute, 60 ?l of EB buffer was placed on the column, incubated for 5 min and centrifuged at 13,000 rcf for 1 min. The Cy3 labeled and Cy5 labeled cDNA for each hybridization were mixed and 1.5 ?l were used for analysis on a ND-1000 spectrophotometer.
Total RNA (~10ng) in a volume of 25 µl was converted into an OmniPlex WTA cDNA library and amplified by WTA PCR using reagents and protocols according to the beta-commercial TransPlex WTA kit (Rubicon Genomics, Ann Arbor, MI). For each sample, a single 10 µl aliquot of the WTA cDNA library was amplified by WTA PCR and products were purified. Four or five 5 ng aliquots of product were subjected to a second WTA PCR amplification in the presence of amino-allyl dUTP for post amplification labeling and products were pooled before proceeding with the hybridization. Yields after all WTA PCR amplifications were between 2 to 5 ?g per reaction. For all WTA amplified cDNA samples with amino-allyl dUTP incorporated (10-20 ?g), nuclease free water was added to 500 ?l, the sample was transferred to a Microcon YM-30 filter, and centrifuged at 13,000 rcf at RT for 12 minutes. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The amount of sample was measured and the volume was adjusted to 5 ?l with nuclease free water. For labeling, 5 ?l of 1 M sodium bicarbonate (pH 9.0) was added and allowed to incubate at RT for 15 min. Nine ?l of anhydrous DMSO were added to Mono Reactive Cy3 and Cy5 dye packs (Amersham, Buckinghamshire, England), mixed thoroughly, and 1 ?l of the proper dye was added to each sample. The labeling mixture was incubated in the dark at RT for 1 hour. The reaction was stopped by the addition of 9 ?l of 4 M Hydroxylamine for 15 min. For each labeling mixture, RNase free water was added to 100 ?l, 500 ?l of PB buffer was added and Cy3 and Cy5 mixtures were added to separate Qiaquick (Qiagen, Valencia, CA) spin columns and centrifuged at 13,000 rcf. Columns were washed twice with 700 ?l of buffer PE, and centrifuged dry at 13,000 rcf for 2 min. To elute, 60 ?l of EB buffer was placed on the column, incubated for 5 min and centrifuged at 13,000 rcf for 1 min. The Cy3 labeled and Cy5 labeled cDNA for each hybridization were mixed and 1.5 ?l were used for analysis on a ND-1000 spectrophotometer.
Hybridization protocol
For hybridization, 40 ug of human Cot-1 DNA (Invitrogen, Carlsbad, CA) was added to 120 ?l of labeled cDNA, the probe was transferred to a Microcon YM-30 filter and centrifuged at 13,000 rcf for 6 min at RT. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The eluted probe volume was adjusted to 18.6 ?l with nuclease free water and the following were added: 4 ?l of yeast tRNA (10 ?g/?l, Invitrogen, Carlsbad, CA), 4.9 ?l of 20X SSC and 0.84 ?l of 10% SDS. The probe was denatured at 100 Co for 3 min and centrifuged at 13,000 rcf for 45 sec. The probe was added directly to the microarray and a cover slip was added. Slides were hybridized overnight in DIE-TECH 1 or 5 slide hybridization chambers in a 65 Co water bath. After hybridization, cover slips were removed by incubation in 2x SSC / 0.05% SDS. Slides were then washed in 2x SSC / 0.05% SDS for 5 min and 0.2x SSC / 0.05% SDS for five minutes. Slides were then washed in 0.2x SSC for 10 sec and centrifuged dry at 500 rpm for 5 min.
Scan protocol
Microarrays were scanned using a GenePix 4000B scanner (Axon Instruments, Union City, CA).
Description
See MIAME checklist, included in the Supplementary Methods, for additional information
Data processing
Images were gridded and spots were quantified using GenePix Pro 4.0 software (Axon Instruments, Union City, CA). For all hybridizations, the log2 normalized Median of Ratios (as described below) was used.
