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Status |
Public on Feb 24, 2012 |
Title |
Spleen_infected day 14_Arizona_replicate 1 |
Sample type |
RNA |
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Source name |
Spleen_infected day 14_Arizona_replicate 1
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Organism |
Haemorhous mexicanus |
Characteristics |
tissue: spleen treatment: infected day 14 population: Arizona replicate: 1
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA extracted from approximately 17 mg of spleen tissue using Qiagen RNeasy miniprep spin columns and followed by DNase digestion of genomic DNA according to the manufacturers’ protocols.
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Label |
Cy5
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Label protocol |
Each pooled House finch RNA sample was prepared for cDNA microarray hybridisation by reverse-transcribing 15µg of total pooled RNA in 30.8 µl reaction volumes containing 1 µl of a mix of oligo dT (dT12-, 13-, 14-, 15-, 16-, 17- and 18-mer; final concentration 5 µg/µl), 6 µl of 5 First Strand Buffer (Invitrogen), 3 µl of 0.1M DTT, 0.8 µl 50 aminoallyl-dUTP/dNTP mix (20mM dATP, 20mM dCTP, 20mM dGTP, 12mM dTTP, 8mM aminoallyl-dUTP), 2.5 µl (500U) of Superscript II Reverse Transcriptase (Invitrogen). Reactions were incubated at 42ºC for 3h. cDNA samples were subsequently hydrolyzed by adding 10 µl of 1M NaOH, 10 µl of 0.5M EDTA and incubated at 65ºC for 15 min. Neutralization was performed by addition of 25 µl 1M HEPES pH 7.5. All samples were labelled using Cy5 dye and the common reference was labelled with Cy3.
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Hybridization protocol |
Immediately prior to hybridisation, we added 0.45 µl 10% SDS to the probe, heated it for 2 min at 100ºC and allowed it to cool down at room temperature for 10 min. The arrays were placed in Corning Microarray hybridisation chambers and a clean Lifterslip (Eerie Scientific) was placed over each array. After injecting the probes under the lifterslips, we added 50 µl 3 SSC to the hybridisation chambers and placed them in a 62ºC water bath for 12-16h. Following hybridisation, the slides were washed in 0.2 SSC with 2% SDS and then in 0.2 SSC. We spun the slides dry by centrifuging them at 1000 rpm for 2 min and scanned using an Axon 4000A microarray scanner (Axon Instruments).
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Scan protocol |
Slides were scanned using an Axon 4000A microarray scanner (Axon Instruments). We used the software package GenePix to yield log base-2 (log2) measurements for mean fluorescence intensities for each dye channel in each spot on the array and to flag low quality spots.
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Data processing |
Normalisation of the raw fluorescence intensities was performed in 3 steps using bioconductor package marray and limma(12) implemented in R language (http://www.r-project.org). First, background adjustment was performed using the normexp method. Second, spatial and print-tip loess normalisation (two dimensional method) were performed to remove spatial and dye biases for each slide. Third, we performed a scale normalisation to control for variation between slides.
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Submission date |
Feb 19, 2012 |
Last update date |
Feb 24, 2012 |
Contact name |
Camille Bonneaud |
E-mail(s) |
bonneaud@dr14.cnrs.fr
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Organization name |
Centre National de la Recherche Scientifique (CNRS)
|
Street address |
Station d'Ecologie Experimentale
|
City |
Moulis |
ZIP/Postal code |
09200 |
Country |
France |
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Platform ID |
GPL15254 |
Series (1) |
GSE35931 |
Innate immunity and the evolution of resistance to an emerging infectious disease in a wild bird |
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