tissue: gill alternative identifier: W_U_LAF_329_HH008_059 Sex: F run: Late run sampling date: 2006-08-31 last tracked zone: 75 last tracked date: 2006-09-11 fate: Upper river mort stock: Lower Adams
Extracted molecule
total RNA
Extraction protocol
Field collected samples were initially stored in a charged liquid nitrogen cryo-shipper and transferred to -80C upon arrival to the lab. Total RNA was purified from individual fish gill using Magmax(TM)-96 for Microarrays Kits (Ambion Inc, Austin, TX, USA) with a Biomek FXP (Beckman-Coulter, Mississauga, ON, Canada) automated liquid-handling instrument. Two gill filaments per fish were homogenized with stainless steel beads in TRI-reagent (Ambion Inc, Austin, TX, USA) on a MM301 mixer mill (Retsch Inc., Newtown, PA, USA). One hundred micro liter aliquots of homogenates were pipetted into 96 well plates and extractions were carried out according to the manufacturer's instructions using the No-Spin Procedure on the Biomek FXP. In the final step, RNA was eluted with RNAase-, DNAase-free water and RNA yield was determined by measuring the A260 of the eluate. Purity was assessed by measuring the A260/A280 ratio of the eluate. Solutions of RNA were stored at -80C until use for cDNA synthesis or quantitative RT-PCR.
Label
Alexa 555
Label protocol
500 nanograms to 5 micrograms of total RNA was amplified 1x using a MessageAmp(TM) II-96 kit (Ambion, TX, USA), performed manually according to manufacturers instructions. Five micrograms of aRNA was reverse transcribed into cDNA and labelled with Alexa dyes using the Invitrogen Indirect Labelling Kit, with modifications from the manufacturer's instructions. Briefly, the cDNA was purified using Zymo-25 Clean-Up columns (Zymo Research, Orange, CA) and eluted using the 2X coupling buffer, supplied by Invitrogen. During dye labelling, samples were processed individually by first adding DMSO, then cDNA to the Alexa dye tube and incubating for 1h at room temperature. All individual (experimental) samples were fluorescently tagged with Alexa 555 and references were labelled with Alexa 647. Samples and references were cleaned up by adding 50 microliters of DNA binding buffer (Zymo) to each Alexa tube and then combining the sample and references for each slide into Zymo-25 Clean-Up columns. The unbound portion of the labelled cDNA was removed by centrifugation at 13,000 rpm/min. The labelled cDNA was washed three times with DNA wash buffer (Zymo) and eluted in 9 microliters of 1X TE buffer. Two microlitres of poly dA was added to the targets, which were then denatured for 10 min at 80C, followed by the addition of 125 microliters of pre-warmed SlideHybe3 buffer (Ambion, TX, USA) before loading into the hybridization chamber in Tecan-HS4800 Pro Hybridization Station (Tecan Trading AG, Switzerland)
Field collected samples were initially stored in a charged liquid nitrogen cryo-shipper and transferred to -80C upon arrival to the lab. Total RNA was purified from individual fish gill using Magmax(TM)-96 for Microarrays Kits (Ambion Inc, Austin, TX, USA) with a Biomek FXP (Beckman-Coulter, Mississauga, ON, Canada) automated liquid-handling instrument. Two gill filaments per fish were homogenized with stainless steel beads in TRI-reagent (Ambion Inc, Austin, TX, USA) on a MM301 mixer mill (Retsch Inc., Newtown, PA, USA). One hundred micro liter aliquots of homogenates were pipetted into 96 well plates and extractions were carried out according to the manufacturer's instructions using the No-Spin Procedure on the Biomek FXP. In the final step, RNA was eluted with RNAase-, DNAase-free water and RNA yield was determined by measuring the A260 of the eluate. Purity was assessed by measuring the A260/A280 ratio of the eluate. Solutions of RNA were stored at -80C until use for cDNA synthesis or quantitative RT-PCR.
Label
Alexa 647
Label protocol
500 nanograms to 5 micrograms of total RNA was amplified 1x using a MessageAmp(TM) II-96 kit (Ambion, TX, USA), performed manually according to manufacturers instructions. Five micrograms of aRNA was reverse transcribed into cDNA and labelled with Alexa dyes using the Invitrogen Indirect Labelling Kit, with modifications from the manufacturer's instructions. Briefly, the cDNA was purified using Zymo-25 Clean-Up columns (Zymo Research, Orange, CA) and eluted using the 2X coupling buffer, supplied by Invitrogen. During dye labelling, samples were processed individually by first adding DMSO, then cDNA to the Alexa dye tube and incubating for 1h at room temperature. All individual (experimental) samples were fluorescently tagged with Alexa 555 and references were labelled with Alexa 647. Samples and references were cleaned up by adding 50 microliters of DNA binding buffer (Zymo) to each Alexa tube and then combining the sample and references for each slide into Zymo-25 Clean-Up columns. The unbound portion of the labelled cDNA was removed by centrifugation at 13,000 rpm/min. The labelled cDNA was washed three times with DNA wash buffer (Zymo) and eluted in 9 microliters of 1X TE buffer. Two microlitres of poly dA was added to the targets, which were then denatured for 10 min at 80C, followed by the addition of 125 microliters of pre-warmed SlideHybe3 buffer (Ambion, TX, USA) before loading into the hybridization chamber in Tecan-HS4800 Pro Hybridization Station (Tecan Trading AG, Switzerland)
Hybridization protocol
Each fish examined in the array experiments was run on a single slide against a reference control that was a pool of all of the fish used in the experiment. Microarray data were expressed in terms of normalized (background corrected) log2 ratios between each fish and the reference control. All slides were processed on Tecan-HS4800 Pro Hybridization Station (Tecan Trading AG, Switzerland). All steps from washing, hybridization, denaturation, and slide drying were carried out automatically.
Scan protocol
Fluorescent images were scanned using a Perkin Elmer ScanArray Express (Perkin Elmer, Boston, MA), adjusting the PMT gain for optimized visualization of each image. The images were quantified using Imagene (BioDiscovery, El Segundo, CA, www.biodiscovery.com).
Data processing
Expression data were managed using a local installation of BASE. BASE was customized slightly to support Imagene two-file formatting. Each slide was normalized in BASE using the print-tip LOESS method. The number of missing values, mean signal-to-noise log-ratio and quality metrics from arrayQualityMetrics and arrayQuality in Bioconductor were used to assess slide quality. Slides were removed from further experimental analysis if two or more plots were flagged on the arrayQualityMetrics report after data normalization, if more than 40% missing spots were identified by the SNR and missing spots report, if there were more than 30% missing spots and an experimentally low SNR value as identified by the SNR and missing spots report, or if the slide had spatial problems as identified by the plots from the arrayQuality package and substantiated by the spatial plots score of the raw microarray data in the arrayQualityMetrics report. Based on these criteria, 6% of slides were typically considered low quality and removed. The data for each retained slide was further processed to remove poor-quality spots. Flagged spots were treated as missing, as were spots with a SNR less than 2. In a set of slides considered a single data set, probes with more than 50% missing values were removed prior to statistical analysis. For procedures such as PCA that require matrices without missing values, missing data were imputed using an average of the existing probe intensities. The submitted data includes all probes and imputed data is not included.
