Comment[ArrayExpressAccession] E-GEOD-61801 MAGE-TAB Version 1.1 Public Release Date 2015-11-04 Investigation Title Comparison between two different Pinus pinaster provenances Comment[Submitted Name] Comparison between two different Pinus pinaster provenances Experiment Description The transcriptome of needles from plants propagated by cuttings and cultured in the same condictions at SERIDA’s greenhouse at Villaviciosa (Asturias, Spain) were analyzed. The cuttings are from two different provenances of Pinus pinaster; Leiria (Portugal) and Tamrabta (Morocco). Their transcriptomes were analyzed using one color microarrays. 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 Cañas Cañas Ávila Cánovas Person First Name Rafael Rafael Concepción Francisco Person Mid Initials A A M Person Email rcanasp@gmail.com Person Affiliation Universidad de Málaga Person Address Biología Molecular y Bioquímica, Universidad de Málaga, Edificio I+D, Campus de Teatinos, Malaga, Spain Person Roles submitter Protocol Name P-GSE61801-1 P-GSE61801-4 P-GSE61801-5 P-GSE61801-2 P-GSE61801-3 P-GSE61801-6 Protocol Description The raw data preprocessing, quantile normalization and differential expressed gene analysis were done with the Limma package for R. ID_REF = VALUE = Normalized signal intensity from spots with foreground significantly different to background Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by NucleoSpin® Gel and PCR Clean-up column purification (Macherey-Nagel, Düren, Germany). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. 1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 mL containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 mL of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the microarrays. They were incubated in SureHyb microarray hybridization chambers with gasket slides (Agilent Technologies) at 65ºC for 17 h in an rotary oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent), 1 minute with 37°C GE Wash buffer 2 (Agilent), 10 s with acetonitrile and 30 s in Agilent's stabilization and drying solution, then air dried. Plants were rooted cuttings of 17 months of age, planted in 2-l pots filled with a mixture of Sphagnum peat (PINSTRUD®) and vermiculite (VERLITE®) of grade 3, with a proportion 4:1 in volume. The entire experiment was performed at SERIDA’s greenhouse at Villaviciosa (Asturias, Spain). Potted plants were ferti-irrigated following the protocol by Majada et al. (2009, 5th Congreso Forestal Español. Ávila, 1–10.). Prior to the assessment, all branches were pruned to trigger the formation of new axillary shoots, mostly deriving from the activation of the proventitious meristems in the extant dwarf shoots. This was intended to be a ‘‘resetting’’ of the plants shoots, minimising the differences due to previous pruning done for cutting collection. Total RNA was isolated following the method of Chang S et al. Plant Mol Biol Rep 1993, 11:113–116 Hybridized slides were scanned at 5 μm resolution and their signal intensities were detected by a GenePix 4100A microarray scanner (Molecular devices, Sunnyvale, CA, USA). Protocol Type normalization data transformation protocol labelling protocol hybridization protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name provenance Experimental Factor Type provenance Comment[SecondaryAccession] GSE61801 Comment[GEOReleaseDate] 2015-11-04 Comment[ArrayExpressSubmissionDate] 2014-09-26 Comment[GEOLastUpdateDate] 2015-11-06 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-61801.sdrf.txt