Investigation Title	tRNA Over-Expression in Breast Cancer	
Comment[Submitted Name]	tRNA Over-Expression in Breast Cancer	
Experimental Design	transcription profiling by array	
Experimental Design Term Source REF	EFO	
Comment[SecondaryAccession]	GSE17945	
Comment[ArrayExpressReleaseDate]	2010-05-22	
Comment[AEMIAMESCORE]	3	
Comment[Publication DOI]	10.1093/nar/gkp787	
Comment[ArrayExpressAccession]	E-GEOD-17945	
Comment[MAGETAB TimeStamp_Version]	2010-08-03 15:12:06 Last Changed Rev: 13058	
Experimental Factor Name	TUMOR TYPE	RECEPTOR STATUS	SOURCE TYPE	TOTAL RNA SOURCE	
Experimental Factor Type	tumor type	receptor status	source type	total rna source	
Experimental Factor Term Source REF	
Person Last Name	Pavon	Gomes	Geslain	Pan	Rich Rosner	Dai	
Person First Name	Mariana	Suzanna	Renaud	Tao	Marsha	Qing	
Person Mid Initials	
Person Email				taopan@uchicago.edu	
Person Phone				773-624-4778	
Person Fax	
Person Address				Biochemistry & Molecular Biology, University of Chicago, 929 East 57th street, Chicago, IL, USA	
Person Affiliation				University of Chicago	
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	2010-05-22	
PubMed ID	19783824	
Publication DOI	19783824	
Publication Author List	Pavon-Eternod M, Gomes S, Geslain R, Dai Q, Rosner MR, Pan T	
Publication Title	tRNA over-expression in breast cancer and functional consequences.	
Publication Status	journal_article	
Publication Status Term Source REF	The MGED Ontology	
Experiment Description	Increased proliferation and elevated levels of protein synthesis are characteristic of transformed and tumor cells. Though components of the translation machinery are often misregulated in cancers, how tRNA plays a role in cancer cells has not been explored. We compare genome-wide tRNA expression in tumorigenic versus non-tumorigenic breast cell lines, as well as tRNA expression in breast tumors versus normal breast tissues. In tumorigenic versus non-tumorigenic cell lines, nuclear-encoded tRNAs increase by up to 3-fold and mitochondrial-encoded tRNAs increase by up to 5-fold. In tumors versus normal breast tissues, both nuclear and mitochondrial-encoded tRNAs increase by up to 10-fold. This tRNA over-expression is selective and coordinates with the properties of cognate amino acids. Nuclear- and mitochondrial-encoded tRNAs exhibit distinct expression patterns, indicating that tRNAs can be used as biomarkers for breast cancer. We analyzed tRNA expression levels in 2 non-tumorigenic breast cell lines, 6 tumorigenic breast cancer cell lines, 3 normal breast tissue samples, and 9 breast tumor samples. We used a non-tumorigenic breast cell line (MCF10A) as a reference sample in all hybridizations. All data is dye-swapped.	
Protocol Name	P-GSE17945-5	P-GSE17945-3	P-GSE17945-4	P-GSE17945-6	P-GSE17945-7	P-GSE17945-8	P-GSE17945-9	P-GSE17945-1	P-GSE17945-2	
Protocol Type	nucleic_acid_extraction	specified_biomaterial_action	grow	labeling	hybridization	image_aquisition	feature_extraction	bioassay_data_transformation	bioassay_data_transformation	
Protocol Description	Cell lines: total RNA for each cell line was obtained at 80-90% confluency using the mirVanaTM miRNA Isolation Kit (Ambion, AM1560). This procedure isolates RNA species as short as 15 nucleotides and is therefore not biased against tRNA. Normal breast: two samples of human breast total RNA were purchased from Ambion (FirstChoice® Human Breast Total RNA, AM6952, lot numbers 0808001 and 0812006). An additional sample of human breast total RNA was purchased from Stratagene (MVP Total RNA Human Breast, 540045, lot number 0380023). Both providers certify small RNA content in total RNA samples and provide relevant donor information. Breast tumors: Breast tumor samples were obtained from the Human Tissue Resource Center (HTRC) at the University of Chicago. Pathology reports were provided for all samples. All breast tumor samples were obtained frozen and embedded in OCT (optimal cutting temperature compound). Following removal of the surrounding OCT and tissue pulverization, total RNA was isolated using TRIzol reagent (Invitrogen, 15596-018). In all cases, total RNA quality was checked on agarose gels.	Deacylation: 0.25µg/µl total RNA premixed with three tRNA standards (E. coli tRNALys, E. coli tRNATyr, and yeast tRNAPhe) at 0.2µM each was incubated in 100mM Tris-HCl (pH 9.0) at 37°C for 30 minutes. The solution was neutralized by the addition of an equal volume of 100mM Na-acetate/acetic acid (pH 4.8) plus 100mM NaCl, followed by ethanol precipitation. Deacylated total RNA was dissolved in water, and its integrity verified by agarose gel electrophoresis.	All cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained according to ATCC recommendations. MCF7 cells were cultured in DMEM medium (Invitrogen, 11965-092) supplemented with 10% FBS and 1% penicillin/streptomycin (P/S). MCF10A cells were cultured in 1:1 (+I factor, Vectro)-DMEM/F12 (Invitrogen, 11330-032) supplemented with 10% FBS, 1% P/S, 5μg/mL insulin, 10ng/mL EGF, and 0.5μg/mL hydrocortisone. All other cell lines were cultured in RPMI 1640 1X medium (Mediatech, 10-040-CV) supplemented with 10% FBS and 1% P/S.	Cy3/Cy5 labeling: after deacylation, tRNA in the total RNA mixture was selectively labeled with either Cy3 or Cy5 fluorophore using an enzymatic ligation method. The ligation reaction relies on an 8-base pair RNA:DNA hybrid helix containing a Cy3 or Cy5 fluorophore pre-attached to the loop and an overhang complementary to the universally conserved 3'CCA nucleotides present in all tRNAs. The ligation reaction was carried out overnight (~16 hours) at 16°C with 1U/µL T4 DNA ligase (USB Corporation, 70042X) and 9µM labeling oligonucleotide.	Hybridization was performed in a GeneMachines Hyb4 at 60°C overnight (16 hours) with 1 to 2.5µg each of Cy3- or Cy5-labeled total RNA as previously described. Slides were washed with 2X SSC / 0.1% SDS, 0.1X SSC / 0.1% SDS, and 0.1X SSC prior to drying and scanning.	Arrays were scanned using a GenePix 4000b scanner and GenePix Pro 6.0 (Axon Instruments). For both Cy3 and Cy5 wavelengths, PMT gain was set at 600 and power at 100%.	Image and data analysis were carried out using GenePix Pro 6.0 and Microsoft Excel. Median Cy5/Cy3 ratios were obtained from all spot replicates. To account for differences in labeling and hybridization efficiencies, the following normalization procedure was used. First, the median Cy5/Cy3 ratio for each probe was divided by the corresponding MCF10A Cy5/Cy3 reference ratio. Second, the obtained value was divided by the averaged Cy5/Cy3 ratio value for the three tRNA standards added as controls (E. coli tRNALys, E. coli tRNATyr, and yeast tRNAPhe).	ID_REF = <br>VALUE = Raw Log Ratio (635/532) from GPR file	ID_REF = <br>VALUE = Raw Log Ratio (532/635) (inversed from GPR file)	
Protocol Parameters	
Protocol Hardware	
Protocol Software	
Protocol Contact	
Protocol Term Source REF							The MGED Ontology	The MGED Ontology	The MGED Ontology	
SDRF File	E-GEOD-17945.sdrf.txt	
Term Source Name	The MGED Ontology		ArrayExpress	EFO	The MGED Ontology	
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php		http://www.ebi.ac.uk/arrayexpress	http://www.ebi.ac.uk/efo/	http://mged.sourceforge.net/ontologies/MGEDontology.php	
Term Source Version