Computational protocol: Transcription profiles of non-immortalized breast cancer cell lines

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Protocol publication

[…] The Atlas Human Cancer 1.2 cDNA expression array (Clontech, CA) is a nylon membrane printed with 200–600 bp fragments of 1176 characterized genes involved in cancer, 9 housekeeping genes and 6 negative controls. RNAs were extracted and labelled with the Atlas pure total RNA labelling system and hybridized according to the manufacturer's instructions.All the cell lines used for the arrays (9 HMECs, 10 MSSMs, 3 N-est and 7 T-est) were probed twice in separate assays. The accuracy of the duplicates was assessed by Pearson's correlation coefficient based on the adjusted intensities of all the genes spotted on the membrane, which ranged from 0.93 to 0.99.Hybridizations with 30 μg of total RNA were performed according to the manufacturer's instructions. The hybridized membranes were exposed to a phosphorimager screen and were read at 100 μm resolution in a Storm Phosphorimaging system (Molecular Dynamics, CA). The scanned images were transformed to TIFF files with a PC bit order and then aligned and analyzed using AtlasImage 2.01 software (Clontech, CA). To average or compare the samples, the adjusted intensity signal was normalized using the global normalization mode featured in the software. We report only (a) those genes with significant (p < 0.01) differential expression of 2-fold or more, and (b) genes that were undefined for all the cell lines belonging to one type of sample, but were detected in other types with a difference at least equivalent to one background (540 units in intensity). (Undefined genes are those for which the intensity was below the signal threshold).The AtlasImage software compares only two samples at a time. When we used it to determine the differences between cell classes, we first averaged the cell lines in the four classes (HMEC, MSSM, N-est and T-est) and then performed the comparisons as instructed by the manufacturer.Statistical analyses (correlation and two-tailed p values) were performed using Microsoft Excel 2000. Further analyses were performed with Significance Analysis of Microarrays (SAM) [], Prediction Analysis for Microarrays (PAM) (Stanford Univ., USA), FatiGO [], Pomelo tool and SVM (Bioinformatics unit, CNIO, Spain []) and GoMiner []. [...] To validate the results of the cDNA array experiments, some of the genes found to be differentially expressed were further examined by real-time PCR in 10 HMECs, 9 MSSMs, 3 N-est and 8 T-est cell lines. Five μg of total RNA (corresponding to about 100 ng of mRNA) were reverse-transcribed with oligo(dT) (SuperScript II system, Invitrogen, CA) in a 20 μl reaction volume, and after 125-fold dilution, 1.25 μl were used for a 40-cycle PCR on an ABI Prism 7900 thermal cycler. The reaction was carried out in a 384-well plate with a QuantiTect SYBR Green PCR kit (Qiagen Inc, CA) at an annealing temperature of 63°C and detection at 2–5°C below the Tm of the product as determined from its dissociation curve. Product size was confirmed by agarose gel electrophoresis. The efficiency of each pair of primers for amplification was determined and expression of each gene relative to G3PDH was assessed by the program Qgene []. Primers were designed using the program PrimerQuest or Primer3, unless otherwise stated. Primer sequences, lengths and Tms of the products are given in .Samples were tested twice in triplicate. Pearson correlation coefficients for the duplicate Q-PCR results ranged between 0.89 and 0.99. […]

Pipeline specifications

Software tools SAM, Babelomics, GoMiner, QGene, PrimerQuest, Primer3
Applications Gene expression microarray analysis, qPCR
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms, Pleural Effusion