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Sample GSM449687 Query DataSets for GSM449687
Status Public on Jan 31, 2010
Title Fall-Winter cDNA pool replicate 1
Sample type RNA
 
Channel 1
Source name pooled Fall total RNA
Organism Osmerus mordax
Characteristics gender: male
tissue: liver
collection month: October
water temperature: 10°C
Treatment protocol Livers were obtained from three male rainbow smelt held at the OSC on October 20th, 2000, when the water temperature was 10ºC and from three male smelt on January 23rd, 2001, when the temperature was 1ºC. Excised livers were treated with RNALater (Ambion) and subsequently stored on dry ice for transport and for the long-term at -80°C
Growth protocol Rainbow smelt were wild-caught in Newfoundland, Canada, and maintained at the Memorial University of Newfoundland Ocean Sciences Centre (OSC) on flow-through water at ambient temperature.
Extracted molecule total RNA
Extraction protocol Liver (30-50 mg) was homogenized in 1 mL TRIzol reagent following the manufacturer’s protocol (Invitrogen) using an RNaseZap (Ambion)-treated Polytron homogenizer (Brinkmann) for 15-20 s on high speed. The tip was rinsed between samples with a series of washes beginning with 0.1% SDS in DEPC water and ending with DEPC water. After air-drying, the total RNA pellet was re-dissolved in nuclease-free water (Sigma-Aldrich) and treated with RNAsecure reagent (Ambion). RNA quality was assessed by electrophoresis on 2% agarose gels in TBE buffer and quantified using a NanoDrop Spectrophotometer (Thermo Fisher).
Label Alexa 546
Label protocol Labelling of cDNAs employing the 3DNA Array 900 chemistry (Genisphere) for two colour analysis using the Alexa Fluor 546 and 647 kits was followed essentially as described by the manufacturers, with minor modifications. For each pool of total RNA, 2 µg of RNA was combined with 1 µL of the fluor-specific RT primer and nuclease-free water to make 6 µL, after denaturing at 80°C for 5 min and cooling on ice, 4.5 µL of a master mix consisting of 5 µL 5x Superscript first strand buffer, 2.5 µL DTT, 1.25 µL Superase-in RNase inhibitor and 1.25 µL Superscript III enzyme (200 units) was added and after mixing and brief centrifugation, the reaction was incubated at 50°C for 2 h. The reaction was stopped by the addition of 1 µL 1 M NaOH and after incubation at 65°C for 10 min to hydrolyse the RNA, the reaction was neutralized by adding 1.2 µL of 2 M Tris-HCl, pH 7.5, for a final reaction volume of 12.7 µL for each fluor.
 
Channel 2
Source name pooled Winter total RNA
Organism Osmerus mordax
Characteristics gender: male
tissue: liver
collection month: January
water temperature: 1°C
Treatment protocol Livers were obtained from three male rainbow smelt held at the OSC on October 20th, 2000, when the water temperature was 10ºC and from three male smelt on January 23rd, 2001, when the temperature was 1ºC. Excised livers were treated with RNALater (Ambion) and subsequently stored on dry ice for transport and for the long-term at -80°C
Growth protocol Rainbow smelt were wild-caught in Newfoundland, Canada, and maintained at the Memorial University of Newfoundland Ocean Sciences Centre (OSC) on flow-through water at ambient temperature.
Extracted molecule total RNA
Extraction protocol Liver (30-50 mg) was homogenized in 1 mL TRIzol reagent following the manufacturer’s protocol (Invitrogen) using an RNaseZap (Ambion)-treated Polytron homogenizer (Brinkmann) for 15-20 s on high speed. The tip was rinsed between samples with a series of washes beginning with 0.1% SDS in DEPC water and ending with DEPC water. After air-drying, the total RNA pellet was re-dissolved in nuclease-free water (Sigma-Aldrich) and treated with RNAsecure reagent (Ambion). RNA quality was assessed by electrophoresis on 2% agarose gels in TBE buffer and quantified using a NanoDrop Spectrophotometer (Thermo Fisher).
Label Alexa 647
Label protocol Labelling of cDNAs employing the 3DNA Array 900 chemistry (Genisphere) for two colour analysis using the Alexa Fluor 546 and 647 kits was followed essentially as described by the manufacturers, with minor modifications. For each pool of total RNA, 2 µg of RNA was combined with 1 µL of the fluor-specific RT primer and nuclease-free water to make 6 µL, after denaturing at 80°C for 5 min and cooling on ice, 4.5 µL of a master mix consisting of 5 µL 5x Superscript first strand buffer, 2.5 µL DTT, 1.25 µL Superase-in RNase inhibitor and 1.25 µL Superscript III enzyme (200 units) was added and after mixing and brief centrifugation, the reaction was incubated at 50°C for 2 h. The reaction was stopped by the addition of 1 µL 1 M NaOH and after incubation at 65°C for 10 min to hydrolyse the RNA, the reaction was neutralized by adding 1.2 µL of 2 M Tris-HCl, pH 7.5, for a final reaction volume of 12.7 µL for each fluor.
 
 
Hybridization protocol Pre-hybridization of the arrays was performed in O-ring sealed aluminum chambers using a solution of final concentration 5X SSC, 0.1% SDS and 0.2% BSA (fraction V) aliquoted beneath a spaced coverslip (Lifterslip) at 49°C for 45 min, after which the arrays were washed in water twice, and then dried by centrifugation. A cDNA hybridization solution consisting of the two cDNA reactions described above, a GFP gridding assist spike, LNA dT blocker and 2X formamide hybridization buffer #7 was introduced (after denaturation at 80°C for 10 min and cooling to 49°C) beneath a fresh Lifterslip placed over the prepared, pre-warmed (to 49°C) arrays, which were then sealed in a humidified chamber for incubation at 49°C overnight.
Scan protocol Microarrays were scanned at 532 and 635 nm using the ScanArray 5000XL (Packard BioChip Technologies) at a resolution of 10 μm with channels balanced based on line scans through representative groups of spots.
Description technical replicate 1 of 3
For each replicate chip (47d1, 48d2 and 49d3) lowess normalized background subtracted median spot intensities are presented as well as Ch2/Ch1 ratios (Winter/Fall) as both arithmetic and log base 2 transformed values.
Data processing Spot intensity values were obtained and normalized using the QuantArray (Packard) microarray analysis software and data were exported to Excel for further analysis. Genes with intensity values ≥ 325 fluorescence units, with a coefficient of variation ≤ 30% within all like-named spots on the array and based upon data from a minimum of two technical replicates, were considered as suitable for inclusion in the analysis. In the set of genes meeting the criteria, those for which the mean January expression value was 2-fold or greater or 0.5-fold or less of their October values were identified as differentially regulated (log base 2 ratios greater than 1 or less than -1).
 
Submission date Sep 03, 2009
Last update date Oct 23, 2009
Contact name Kathryn Vanya Ewart
E-mail(s) vanya.ewart@nrc-cnrc.gc.ca
Phone (902) 426-0636
Fax (902) 426-9413
URL http://www.nrc-cnrc.gc.ca/eng/ibp/imb.html
Organization name NRC Institute for Marine Biosciences
Department Marine Bioactives Group
Street address 1411 Oxford St.
City Halifax
State/province NS
ZIP/Postal code B3H 3Z1
Country Canada
 
Platform ID GPL6260
Series (1)
GSE17961 Winter and fall gene expression in rainbow smelt liver

Data table header descriptions
ID_REF
VALUE log base 2 transformed Ch2/Ch1 ratios (Winter/Fall)
47d1 Ch1 N (Median-B)
47d1 Ch2 N (Median-B)
47d1 Ch2/Ch1 N Ratio of Medians

Data table
ID_REF VALUE 47d1 Ch1 N (Median-B) 47d1 Ch2 N (Median-B) 47d1 Ch2/Ch1 N Ratio of Medians
23 0.131672633 879 963 1.09556314
24 0.667841234 10773 17115 1.588693957
26 0.437137874 664 899 1.353915663
27 0.63638215 3050 4741 1.55442623
36 1.549258439 900 2634 2.926666667
46 -1.207473703 1411 611 0.433026223
63 -0.62502199 566 367 0.648409894
64 -0.324677559 16807 13420 0.798476825
80 -0.662965013 627 396 0.631578947
86 0.880708047 1783 3283 1.841278744
89 -0.986824611 654 330 0.504587156
95 0.968955059 869 1701 1.957422325
97 -0.549965218 25331 17302 0.683036595
98 0.992768431 400 796 1.99
100 -0.001647584 6133 6126 0.998858634
104 1.082584802 1307 2768 2.117827085
106 -1.014277158 4062 2011 0.495076317
124 1.093714435 447 954 2.134228188
129 -0.840403757 1137 635 0.558487247
130 -1.820206537 1635 463 0.283180428

Total number of rows: 1915

Table truncated, full table size 69 Kbytes.




Supplementary file Size Download File type/resource
GSM449687_47d1.txt.gz 2.2 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data are available on Series record

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