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Sample GSM449688 Query DataSets for GSM449688
Status Public on Jan 31, 2010
Title Fall-Winter cDNA pool replicate 2
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 2 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)
48d2 Ch1 N (Median-B)
48d2 Ch2 N (Median-B)
48d2 Ch2/Ch1 N Ratio of Medians

Data table
ID_REF VALUE 48d2 Ch1 N (Median-B) 48d2 Ch2 N (Median-B) 48d2 Ch2/Ch1 N Ratio of Medians
23 -0.29432484 1333 1087 0.815453863
24 1.26992587 7327 17669 2.411491743
26 0.24239464 880 1041 1.182954545
27 0.853239346 3510 6341 1.806552707
36 0.259242827 1778 2128 1.196850394
46 0.057930111 659 686 1.040971168
63 -1.382529342 863 331 0.383545771
64 -0.290014498 24623 20139 0.817893839
80 0.242801753 371 439 1.18328841
86 -0.004447863 2274 2267 0.996921724
89 -0.839327312 730 408 0.55890411
95 0.07933737 796 841 1.056532663
97 -0.189825163 37311 32711 0.876711962
98 -0.434987089 534 395 0.739700375
100 0.221083931 9770 11388 1.165609007
104
106 -0.680819341 6938 4328 0.623810897
124 0.345348339 1442 1832 1.270457698
129 -0.645060722 1348 862 0.639465875
130 -0.715666192 1143 696 0.608923885

Total number of rows: 1915

Table truncated, full table size 73 Kbytes.




Supplementary file Size Download File type/resource
GSM449688_48d2.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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