title: Thalassiosira pseudonana NimbleGen 36mer-oligo gene-specific array
status: Public on Jan 05 2008
contact.city: Seattle
contact.first: E.
contact.middle: Virginia
contact.last: Armbrust
contact.email: armbrust@uw.edu
contact.state: WA
contact.address: Ben Hall IRB, 616 NE Northlake Pl, Room 306
contact.department: Oceanography
contact.country: USA
contact.institute: University of Washington
organism: Thalassiosira pseudonana CCMP1335
description: Only probes matching Thalassiosira pseudonana genome version 3 are listed.  Any probes matching more than 4 duplicate positions in Thalassiosira pseudonana genome version 3 are not represented here.
manufacture_protocol: 1.  Gene-specific array design:  A gene-specific array was designed with 176,320 36-mer genomic probes that were selected from the preliminary set of 15,906 transcribed units (TUs) based on the tiling arrays, existing EST data and the version 2 JGI models. The design included up to 68 probes from each gene or transcribed regions. Each array also included ca. 500 random duplicated probes for normalization and noise estimation purposes. The location of the probes on each array was randomized in order to eliminate any positional bias in the synthesis and hybridization.
  2. Synthesis: Six complete sets (in quadruplicate for each growth condition (biological replicates)) of the gene-specific microarrays were synthesized for hybridization with labeled cDNA from the control, low silicon, nitrogen, iron, temperature and alkaline pH. Microarrays were fabricated using a modified Maskless Array Synthesizer (MAS) as described previously (Sing-Gasson et al. 1999, Nuwaysir et al. 2002) on SuperClean (ArrayIT) glass substrates functionalized with N-(3-Triethoxysylylpropyl)-4-hydroxy-butryamide (Gelest Inc). Delivery of standard DNA synthesis reagents (Proligo) and NPPOC phosphoramidite (Nimblegen) was performed via an Expedite DNA synthesizer.
  3. Array Processing: After synthesis, microarrays were deprotected by immersion in EDA/EtOH (1:1 vol) for two hours at room temperature.  Arrays were then washed in water, dried with argon, and stored desiccated prior to hybridization.  Hybridization was performed with 10ug of fragmented cRNA in 300ul in the presence of 100mM MES, 1M Na, 20mM EDTA and 0.01% tween20 for 17 hours at 45C.  After hybridization, arrays were washed with a non stringent wash buffer (NSWB) (6X SSPE and 0.01% Tween 20) followed by incubation with a stringent wash buffer (100mM MES, 0.1M Na+, 0.01% Tween 20) for 30 minutes at 45C.  Arrays were stained with streptavidin-cy3 (Amersham) for 25 minutes at room temperature, washed with NSWB, rinsed with Final Wash Buffer (Nimblegen) and dried under argon. Arrays were scanned using an Arrayworx scanner (Applied Precision).  Array feature intensity was extracted from images using in-house software.