Comment[ArrayExpressAccession]	E-GEOD-57023
MAGE-TAB Version	1.1														
Public Release Date	2014-08-04
Investigation Title	Identification of microRNAs specific for high producer CHO cell lines														
Comment[Submitted Name]	Identification of microRNAs specific for high producer CHO cell lines
Experiment Description	Mature microRNA levels were analyzed in 4 producing CHO cell lines expressing 2 structurally different recombinant proteins at low and high level as well as in 1 non-producing CHO cell line. The goal was to identify microRNAs that are involved in heterologous protein formation and secretion. 5 recombinant CHO cell lines. 3 biological replicates each. All samples were hybridized against a common reference (pool of all total RNA samples).														
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl													
Person Last Name	Hackl	Maccani	Hackl	Leitner	Steinfellner	Graf	Tatto	Karbiener	Scheideler	Grillari	Mattanovich	Kunert	Borth	Grabherr	Ernst
Person First Name	Matthias	Andreas	Matthias	Christian	Willibald	Alexandra	Nadine	Michael	Marcel	Johannes	Diethard	Renate	Nicole	Reingard	Wolfgang
Person Mid Initials						B	E								
Person Email	matthias.hackl@boku.ac.at														
Person Affiliation	University of Natural Resources and Life Sciences Vienna														
Person Phone	+43 1 47654 6803														
Person Address	Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria														
Person Roles	submitter														
Protocol Name	P-GSE57023-1	P-GSE57023-5	P-GSE57023-6	P-GSE57023-2	P-GSE57023-3	P-GSE57023-4	P-GSE57023-7								
Protocol Description	Feature extraction was conducted using the GenePix software (Molecular Devices, Sunnyvale, CA, USA). The LIMMA package of R/Bioconductor (Smyth 2004) was applied for background correction, normalization and statistical analysis as previously described (Hackl et al. 2010). Normexp background correction and Global Loess normalization were performed and log2-fold changes of miRNA expression for each sample were calculated against the common reference. ID_REF =  VALUE = Global lowess normalized log2 ratio between test sample and common reference pool (log2 test/reference), signal intensity > average background intensity + 2 * standard deviation background intensity	End-labeling of 800 ng of total RNA was conducted using the Exiqon Power Labeling Kit (Exiqon, Denmark) together with synthetic spike-in controls according to the manufacturerM-bM-^@M-^Ys instructions.	The arrays were hybridized for 16 h at 56M-0C followed by automated washing and drying with nitrogen using a Tecan HS 400 hybridization station (Tecan, Switzerland).	No treatments were performed	Steady-state cultivations were conducted in 800 mL cell culture bioreactors (DS0700TPSS, DASGIP, Germany). The inocula were expanded in spinner flasks starting from the working cell bank. Exponentially growing cells from passage six were used for inoculation. The initial cell concentration was 2.5E5 cells/mL. The cultures were maintained at 37M-0C, pH 7.0, 30% dissolved oxygen and an agitation speed of 80 rpm. The medium was composed of DMEM without glucose and HamM-bM-^@M-^Ys F12 (1:1) supplemented with 4 mM L-glutamine, 0.25% soy peptone (Quest International, The Netherlands), 0.1% Pluronic F68 and a protein free supplement (Polymun Scientific, Austria). After 3 days of batch cultivation, the process was switched to continuous operation for 11 days. Fresh medium was supplied at a constant flow rate to maintain a dilution rate D of 0.5 1/d. The working volume was kept constant at 400 mL using a DASGIP level sensor. Total RNA for microarray analysis was extracted at the end of the process (steady-state).	Total RNA samples were isolated from 5E6 cells using the Ambion TRI Reagent (Life Technologies, CA, USA) according to the manufacturerM-bM-^@M-^Ys instructions using chloroform for extraction. Yield and purity were determined using the NanoDrop 1000 sprectrophotometer (Thermo Fisher Scientific, MA, USA). Only total RNA samples with an A260/A280 ratio between 1.8 and 2.0 and an A260/A230 ratio >2.0 were used in this study. The integrity of the RNA samples was analyzed using the Agilent 2100 Bioanalyser together with the RNA 6000 Nano LabChip kit (Agilent, CA, USA). The RNA integrity number (RIN) was M-bM-^IM-%9.9 for all samples which indicates a very high sample quality.	Slides were then scanned at 10 M-5m resolution and auto-gain settings using a Roche Nimblegen MS200 scanner (Roche, Germany).								
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol								
Experimental Factor Name	PRODUCTIVITY	RECOMBINANT PROTEIN													
Experimental Factor Type	productivity	recombinant protein													
Comment[SecondaryAccession]	GSE57023														
Comment[GEOReleaseDate]	2014-08-04														
Comment[ArrayExpressSubmissionDate]	2014-04-23														
Comment[GEOLastUpdateDate]	2014-08-05														
Comment[AEExperimentType]	transcription profiling by array														
SDRF File	E-GEOD-57023.sdrf.txt