title: EBT_UA_Enchytraeus albidus_version2
status: Public on Dec 28 2010
contact.city: Aveiro
contact.first: Sara
contact.last: Novais
contact.email: sara.novais@ua.pt
contact.address: Campus de Santiago, Department of Biology, University of Aveiro
contact.department: Biology
contact.country: Portugal
contact.institute: CESAM & University of Aveiro
organism: Enchytraeus albidus
description: Platform is based on a non-model organism, so information is unavailable for some of the genes from this library. Of the 480 genes, 348 have been sequenced and are listed in the Platform table with the respective accession number from GenBank.
manufacture_protocol: The microarray is composed of a normalised cDNA library that followed the protocol of Shagin (Shagin et al., 2002). A pooled RNA sample - containing RNA aliquots from the individuals exposed to all the different exposure conditions - was used for the construction of the normalized cDNA library. The exposures included different concentrations of two chemical compounds and different soil types. In general, the differentially expressed genes were ligated in a TA-vector system using the pGEM-T easy vector (Promega, USA). Escherichia coli competent cells (JM109, Promega) were transformed through heat shock. Clones were amplified with vector specific primers (T7 and SP6 primers, Promega) and purified by an exosap reaction. In preparation for spotting, cDNA-inserts of the clones were, amplified using adaptor specific primers (nested primers, Clontech), purified by Montage PCR Plate (Millipore, USA) and loaded into 384-well plates (Genetix, UK) in 50% dimethylsulfoxide at ~150 ng/µl. A MicroGrid Compact spotter (Biorobotics) was used to spot each clone in triplicate onto aminosilane coated glass slides (Generoma microarray slides; Asper Biotech, Estonia). In addition, a set of artificial genes control genes (Lucidea Universal Scorecard, Amersham Biosciences, UK) was spotted in 20 replicates spread over each array: 10 calibration controls, 8 ratio controls and 2 negative controls. After rehydration and drying, the slides were fixated by UV-radiation at 300mJ (UV Stratalinker 2400; Stratagene, USA).