Investigation Title Transcription profiling of human breast cancer samples kept at room temperature for different lengths of time before freezing Comment[Submitted Name] BIOSPECIMENS HANDLING Experimental Design RNA_stability_design reference_design time_series_design co-expression_design transcription profiling by array Experimental Design Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology EFO Comment[SecondaryAccession] Comment[ArrayExpressReleaseDate] 2009-12-02 Comment[AEMIAMESCORE] 4 Comment[ArrayExpressAccession] E-MEXP-2035 Comment[MAGETAB TimeStamp_Version] 2010-09-07 00:21:37 Last Changed Rev: 13833 Experimental Factor Name TIME Experimental Factor Type time Experimental Factor Term Source REF Person Last Name Callari Person First Name Maurizio Person Mid Initials Person Email maurizio.callari@istitutotumori.mi.it Person Phone +39 0223903053 Person Fax Person Address Dipartimento Oncologia Sperimentale, Via Venezian, 1, Milan, Italy, 20133, Italy Person Affiliation Istituto Nazionale dei Tumori Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2009-12-02 PubMed ID Publication DOI Publication Author List De Cecco L, Musella V, Veneroni S, Cappelletti V, Bongarzone I, Callari M, Valeri B, Pierotti MA, Daidone MG Publication Title Impact of biospecimens handling on biomarker research in breast cancer Publication Status Publication Status Term Source REF Experiment Description In order to gain awareness of the gene expression artifacts linked to improper tissue handling also for this particular type of neoplasia, we have investigated gene expression profiles in a set of primary breast tumors, grossly subdivided into 4 aliquots and kept at room temperature for 0, 2, 6 and 24 hours after resection and before snap-freezing in liquid nitrogen. Protocol Name P-MTAB-3665 P-MTAB-3663 P-MTAB-3664 P-MTAB-3667 Protocol Type nucleic_acid_extraction specified_biomaterial_action labeling hybridization Protocol Description Total RNA was extracted using Trizol (Life Technologies, Frederick, MD, USA) following the manufacturer’s instructions. Integrity ofthe RNA was assessed by agarose gel electrophoresis after DNase I and clean-up treatment with RNaesy mini kit (Qiagen, Valencia, CA, USA).
(Parameters: Extracted product = genomic_DNA, Amplification = none) Histologically confirmed primary breast tumors were obtained from the Tissue Bank of the Fondazione IRCCS Istituto Nazionale Tumori between October 2006 and March 2007. Each tissue sample was divided into 4 aliquots. One was immediately frozen while the remaining three were frozen after 2, 6 and 24 hours at room temperature. Probes were both directly (Cy-dCTP, Amersham, GE) and indirectly (Array900 detection kit, Genisphere). Total RNA was reverse transcribed using modified oligo-dT primers containing the specific 3DNA capture sequences following the manufacturer’s instructions. Probe labeling reaction was carried out in a final volume of 40 ul (1X first-strand buffer; 0.01 M DTT, 0.1 mM dATP, dGTP, dTTP, 6.25 uM dCTP; 0.33 Cy-dCTP; 20 U RNase inhibitor from human placenta;300 U SuperScript II reverse transcriptase (Life Technologies, Fredrick, MA). Samples were incubated at 42°C for 2 h and the reactions stopped by addition of 4 ul of 0.5 M EDTA, pH 8. Starting RNA template was removed by alkali hydrolysis adding 4 ul of 0.5 M NaOH, followed by incubation at 70°C for 15 min and finally neutralization with 4 ul of 0.5 M HCl. Unincorporated nucleotides were removed from labeled probes using Microcon YM-50 columns (Millipore, Bedford, MA). Tumour samples were Cy5 labeled, whereas the Universal human Reference RNA was Cy3 labeled.
(Parameters: Amplification = none, Mass unit = Micro gram) The labeled cDNA probes were annealed with 1.5 ml of the specific 3DNA capture reagent (Array900 detection kit, Genisphere) for 16 h at 40C. Hybridization was carried out in a hybridization station(Genomic Solutions, Ann Arbor, MI, USA) for 16 h at 42C.
(Parameters: Chamber type = OTHER: type 7 star slides (Amersham Biosciences, Amersham, UK), Quantity of label target used = 10, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 42) Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF SDRF File E-MEXP-2035.sdrf.txt Term Source Name EFO ncbitax NCI_thesaurus The MGED Ontology ArrayExpress The MGED Ontology EFO Term Source File http://www.ebi.ac.uk/efo/ http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html ncithesaurus.obo.alt http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ Term Source Version