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Status |
Public on Jan 31, 2010 |
Title |
Fall-Winter cDNA pool replicate 2 |
Sample type |
RNA |
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|
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.
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|
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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.
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|
|
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).
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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
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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 |
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