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Series GSE16

Query DataSets for GSE16

Status

Public on Feb 12, 2002

Title

Microarray-based CGH in human cancer cell lines

Organism

Homo sapiens

Experiment type

Genome variation profiling by genome tiling array

Summary

This series represents the data set from the paper Assembly of microarrays for genome-wide measurement of DNA copy number appearing in Nat Genet 2001 Nov;29(3):263-4 and it's web supplement.
Specimen procurement, labeling, hybridization and imaging protocols from Nat Genet 2001 Nov;29(3):263-4 web supplement:
Specimens. We obtained GM03563, GM00143, GM05296, GM07408, GM01750, GM03134, GM13330, GM03576, GM01535, GM07081, GM02948, GM04435, GM10315, GM13031 and GM01524 cell strains from the NIGMS Human Genetics Cell Repository (Coriell Institute for Medical Research), and cultured them under the recommended conditions. We cultured cell lines BT474, HCT116, T47D, MPE600, COLO320, SW837, MDA-MB-231, MDA-MB-453 and HT29 under the recommended conditions. To isolate DNA from confluent cell cultures, we collected cells from T75 flasks and then incubated them overnight at 55 C in a 3 ml solution containing 0.01 M Tris, pH 7.5, 0.001 M EDTA, pH 8, 0.5% SDS and 0.1 g/l proteinase K. We added 1 ml of saturated NaCl solution and 10 ml of ethanol to the cell lysate and collected the DNA by spooling. We removed excess liquid and then dissolved the DNA in 1 ml of H2O. We isolated DNA from breast tumor specimens as described previously.
DNA labeling. We labeled DNA by nick translation or random priming. For nick translation, we used a 400 l reaction containing 10 g DNA, 50 mM Tris, pH 7.6, 5 mM MgCl2, 0.02 mM each dATP, dTTP and dGTP, 0.2 mM Cy3- or Cy5-dCTP (Amersham), 0.2 U DNA Polymerase I (Gibco BRL), and 30 l DNAseI/PolI enzyme mix (Gibco BRL). We incubated the reaction at 15 C for 60 min after which we stopped the reaction by incubation at 70 C for 10 min. We removed unincorporated nucleotides using a Sephadex G-50 spin column. We labeled genomic DNA by random priming in a 100 l reaction containing 0.003 - 0.6 g DNA, 1x random primers solution (BioPrime DNA Labeling System, Gibco BRL), 1 mM Tris, pH 7.6, 0.1 mM EDTA, 0.2 mM each of dATP, dTTP and dGTP, 0.1 mM dCTP, 0.4 mM Cy3 or Cy5-dCTP (Amersham) and 160 U Klenow fragment (BioPrime DNA Labeling System, Gibco BRL). We incubated the DNA with the random primers solution at 100 C for 10 min in a total volume of 84 l, prior to adding the other reagents and then incubated the 100 l reaction overnight at 37 C. We removed unincorporated nucleotides using a Sephadex G-50 column.
Hybridization. We combined test and reference DNAs (~2 g of input genomic DNA for nick translation or ~0.6 g of input genomic DNA for random prime labeling) with Cot-1 DNA (80-100 g; Gibco BRL) and precipitated them with ethanol. We collected the precipitate by centrifugation and allowed the precipitate to dry in air for 10 min before re-dissolving it in a 50 l hybridization mixture containing 50% formamide, 2 x SSC, 10% dextran sulfate, 4% SDS and 500 g yeast tRNA, pH 7. We incubated the hybridization mixture at 70 C for 10-15 min to denature the DNA and subsequently continued incubation at 37 C for 60 min to allow blocking of repetitive sequences. We applied a ring of rubber cement closely around the array to form a well, into which we added 50 l of slide blocking solution containing 500 g salmon sperm DNA in 50% formamide, 2 x SSC, 10% dextran sulfate and 4% SDS, pH 7. We created an airtight hybridization chamber by placing a silicone gasket (PGC Scientific) around the array and rubber cement ring, placing a slide on top of it and clamping with binder clips. After a 30 min incubation at room temperature, we opened the chamber, removed approximately three-quarters of the blocking solution, added the denatured and re-annealed hybridization mixture and then re-sealed the chamber. We placed the arrays on a slowly rocking table (~1 rpm) at 37 C to allow hybridization to occur over 16-72 h. After hybridization, we rinsed off the excess hybridization fluid with PN buffer (PN: 0.1 M sodium phosphate, 0.1% nonidet P40, pH 8), then washed once in 50% formamide, 2 x SSC, pH 7 at 45 C for 15 min, and finally in PN buffer at room temperature for 15 min. After draining excess liquid from the arrays, we mounted them in a solution containing 90% glycerol, 10% PBS and 1 M DAPI, and then sealed them with a cover slip. We drained excess glycerol-DAPI solution onto a paper tissue.
Imaging and analysis. We acquired 16 bit 1024x1024 pixel DAPI, Cy3 and Cy5 images using a custom built CCD camera system, although we have also used commercial laser scanners for this purpose. We used ?UCSF SPOT? software (A.N.J. manuscript submitted) to automatically segment the spots based on the DAPI images, perform local background correction and to calculate various measurement parameters, including log2ratios of the total integrated Cy3 and Cy5 intensities for each spot. We used a second custom program SPROC to associate clone identities and a mapping information file with each spot so that the data could be plotted relative to the position of the BACs on the September, 2000 freeze of the draft human genome sequence (http://genome.ucsc.edu). SPROC also implements a filtering procedure to reject data based on a number of criteria, including low reference/DAPI signal intensity and low correlation of the Cy3 and Cy5 intensities with a spot. The SPROC output consists of averaged ratios of the triplicate spots for each clone, standard deviations of the triplicates and plotting position for each clone on the array, as well as other clone information stored in the database, such as STS content (Tables A and B). We edited the data files to remove ratios on clones for which only one of the triplicates remained after SPROC analysis and/or the standard deviation of the log2ratios of the triplicates was > 0.2.
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