Sample Extraction Protocol Other: Total RNA was isolated with RNeasy minikits (Qiagen, Germantown, MD) and amplified into anti-sense RNA as previously described (Wang,E., Miller,L., Ohnmacht,G.A., Liu,E. & Marincola,F.M.). High fidelity mRNA amplification for gene profiling using cDNA microarrays. Nature Biotech 17, 457-459; 2000).Quality of RNA was tested by Agilent technologies and spectophotometry (OD). For microarray analysis test samples were labeled with Cy5-dUTP (Amersham, Piscataway, NJ) while the reference sample (pooled normal donor PBMC) was labeled with Cy3-dUTP. Test-reference sample pairs were mixed and co-hybridized to 17K-cDNA microarrays generated in our lab (gal file http://nciarray.nci.nih.gov/gal_files/gal_custom_current.shtml, Hs-CCDTM17.5k-1px.gal
Label
cy5
Label protocol
Cy5 Sample Labeling Protocol Other: Target labeling by reverse transcription: 4ul First strand buffer 1ul dN6 primer (8ug/ul; Boehringer Mannheim Cat# 1034731 resuspended in 250ul DEPC H2O) 2ul 10X low T - dNTP (5mM A, C and GTP, 2mM dTTP) 2ul Cy-dUTP (1mM Cy5) 2ul 0.1 M DTT 1ul RNasin 3-6ug amplified mRNA in 7 ul DEPC H2O Mix well and heat to 65C for 5min then cool down to 42C. Add 1.5ul SSII and Incubate for 90 min at 42C. Add 2.5ul 500mM EDTA and heat to 65C for 1min. Add 5ul 1M NaOH and incubate at 65C for 15min to hydrolyze RNA. Add 12.5ul 1M Tris immediately to neutralize the pH. Add 35ul of 1xTE. Target clean up: Prepare Bio-6 column and run target solution through it. Collect flow through and add 200ul 1xTE. Concentrate target to ~20ul using a microcon YM-30 column (Millipore Cat# 42410).
Sample Extraction Protocol Other: Total RNA was isolated with RNeasy minikits (Qiagen, Germantown, MD) and amplified into anti-sense RNA as previously described (Wang,E., Miller,L., Ohnmacht,G.A., Liu,E. & Marincola,F.M.). High fidelity mRNA amplification for gene profiling using cDNA microarrays. Nature Biotech 17, 457-459; 2000).Quality of RNA was tested by Agilent technologies and spectophotometry (OD). For microarray analysis test samples were labeled with Cy5-dUTP (Amersham, Piscataway, NJ) while the reference sample (pooled normal donor PBMC) was labeled with Cy3-dUTP. Test-reference sample pairs were mixed and co-hybridized to 17K-cDNA microarrays generated in our lab (gal file http://nciarray.nci.nih.gov/gal_files/gal_custom_current.shtml, Hs-CCDTM17.5k-1px.gal
Label
cy3
Label protocol
Cy3 Sample Labeling Protocol Other: Target labeling by reverse transcription: 4ul First strand buffer 1ul dN6 primer (8ug/ul; Boehringer Mannheim Cat# 1034731 resuspended in 250ul DEPC H2O) 2ul 10X low T - dNTP (5mM A, C and GTP, 2mM dTTP) 2ul Cy-dUTP (1mM Cy3) 2ul 0.1 M DTT 1ul RNasin 3-6ug amplified mRNA in 7 ul DEPC H2O Mix well and heat to 65C for 5min then cool down to 42C. Add 1.5ul SSII and Incubate for 90 min at 42C. Add 2.5ul 500mM EDTA and heat to 65C for 1min. Add 5ul 1M NaOH and incubate at 65C for 15min to hydrolyze RNA. Add 12.5ul 1M Tris immediately to neutralize the pH. Add 35ul of 1xTE. Target clean up: Prepare Bio-6 column and run target solution through it. Collect flow through and add 200ul 1xTE. Concentrate target to ~20ul using a microcon YM-30 column (Millipore Cat# 42410).
Hybridization protocol
Sample Hybrization Protocol Wash procedure: Washing: 1. Wash with 2x SSC + 0.1% SDS to remove coverslip. 2. Wash with 1x SSC for 1 min. 3. Wash with 0.2x SSC for 1 min. 4. Wash with 0.05x SSC for 10-20 seconds. 5. Centrifuge slide at 80-100 g for 3 min. Other: Hybridization: Combine Cy3 and Cy5 labeled target (adjust the color to purple) and complete dry the sample by speed vacuum . Add 37ul of the following mixture to the dried target (for 20x40mm slide): 1ul 50x Denhardt’s blocking solution (Sigma Cat# 2532) 1ul poly dA (8mg/ml Pharmacia Cat# 27-7988-01) 1ul yeast tRNA (4mg/ml Sigma Cat# R8759) 10ul Human Cot I DNA (1mg/ml Gibco BRL Cat# 15279-011) 3ul 20X SSC 1ul 10% SDS 20ul of DEPC H2O Heat for 2 min at 99oC and Cool to RT. Apply target mixture to array slide, add coverslip, place in humidified hyb chamber, and hybridize at 65oC over night.
BRBArrayTools Calculation Method: Fluorescence intensities generated by Cy5 and Cy3 probes hybridized onto the microarray slides were scanned were scanned on a GenePix 4000 (Axon Instruments, Union City, CA) at variable PMT voltage to obtain maximal signal intensities with < 1% probe saturation. Data were uploaded to National Institute of Health, NCI/CCR µArray database (http://nciarray.nci.nih.gov). Raw data were retrieved in BRB-ArrayTools(http://linus.nci.nih.gov/BRBArrayTools.html) format for analysis and normalization. Following filter criteria was applied to the data set: 1. spots were excluded if intensity < 100, and spot size less than 25µm 2. genes were filtered out if >50% of experiment with missing data values. 3. log2 transformed data were normalized using lowess smoother, and intensity ratios were truncated if greater than 64. Transcriptional data were process by averaging results in replicate experiments for the same genes.Hierchical clustering analysis was performed on BRBArray tools using centered correlation as similarity metric and centroid linkage method: Cluster 3:0. Heat maps were generated using Eisen’s TreView Software.
