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Sample GSM117520 Query DataSets for GSM117520
Status Public on Aug 09, 2006
Title Chicken day six liver, Vehicle Control rep4 vs. reference RNA pool
Sample type RNA
 
Channel 1
Source name Chicken liver, day six of treatment
Organism Gallus gallus
Characteristics Chicken liver, day six of treatment
Biomaterial provider Moyer's Hatchery (Quakertown, PA)
Treatment protocol At 29 days of age, broiler cockerels (Ross x Cobb) were given a subcutaneous implant of an osmostic minipump (Alzet, model 2001; ALZA, Mountain View, CA) to continuously release hormone for seven days at a rate of 1.0 microliter/hour. Birds received a vehicle (50% DMSO and 50% propylene glycol) as control (VC), exogenous corticosterone (600 micrograms CS/kg/day), exogenous L-tri-idothyronine (192 micrograms T3/kg/day), or both CS and T3. After six days of treatment, liver tissue was excised immediately, snap frozen in liquid nitrogen, and stored in -80 degrees C until RNA isolation.
Growth protocol Chicks were fed a broiler starter ration and raised in a controlled-environment animal room under a 20-h light:4-h darkness photoperiod in a heated battery-brooder until 3 weeks of age. At 3 weeks of age, 72 birds of uniform body weight (within 1 SD) were randomly assigned to wire cages within two identical controlled-temperature rooms with three birds per pen. Rooms were maintained under the same 20L:4D light/dark cycle throughout the duration of the experiment. The birds were provided with water and a commercial grower/finisher ration ad libitum until the end of the experiment.
Extracted molecule total RNA
Extraction protocol Total RNA from liver samples of birds that received six days of treatment was isolated using a RNeasy Midi kit (Qiagen, Valencia, CA) following the manufacturer's protocol with some modifications. The quantity of extracted total RNA was determined using a NanoDrop spectrophotometer (Wilmington, DE), and the quality of extracted total RNA was examined by microcapillary electrophoresis on a BioAnalyzer 2100 (Agilent, Wilmington, DE), where the rRNA ratio (28S/18S) was observed for integrity.
Label Alex Fluor 555
Label protocol Chicken cDNA targets were synthesized with the SuperScript? Plus Indirect cDNA Labeling System (Invitrogen). First, total RNA samples (20 micrograms) were reverse transcribed using an anchored oligo(dT)20 primer, amino-modified dNTPs, and SuperScript III Reverse Transcriptase in 30 microliter volume. Next, the RNA template was destroyed by incubation with sodium hydroxide at 70 degrees C. First strand cDNA was purified with a Low-Elution Volume Spin Cartridge and then labeled with Alexa Fluor 555 (green) in a fluorsecent dye coupling reaction. Dye-incorporated cDNAs were purified to remove un-reacted dye and analyzed for their labeling efficiency with a NanoDrop spectrophotometer.
 
Channel 2
Source name Reference pool of hepatic RNA from five-week-old control chickens
Organism Gallus gallus
Characteristics Reference pool of hepatic RNA from five-week-old control chickens
Extracted molecule total RNA
Extraction protocol Extraction was conducted as described above for channel 1. Equal amounts of total RNA from six control birds were combined to form a reference RNA pool.
Label Alexa Fluor 647
Label protocol 23 aliquots of the reference RNA were labelled with Alexa Fluor 647 (red) as decribed above for channel 1. A pool was made of all Alexa Fluor 647 -labeled cDNA to be split among the 23 slides in the reference hybridization experiment.
 
 
Hybridization protocol After pre-hybridization of the microarray slides in blocking reagent (1% BSA, 5 x SSC, 0.2% SDS), each slide was hybridized with a sample labeled with Alexa Fluor 555 (green) and a reference RNA sample labeled with Alexa Fluor 647 (red) in a reference hybridization design. Slides were hybridized overnight in a 42 degree C water bath under a light-tight box. On the following day, slides were sequentially washed with 1 x SSC, 0.2% SDS, and 0.5% DTT at 55 degrees C for 10 min, then 0.1 x SSC, 0.2% SDS, and 0.5% DTT for 5 min at room temperature, and finally 0.1 x SSC and 0.5% DTT for 1 min at room temperature, with nitrogen gas bubbled through each of the washes to prevent interference of ozone. The slides were subsequently rinsed in dH2O and dried by centrifugation. Before scanning, slides were stored in individual 50 ml tubes covered in aluminum foil and filled with nitrogen gas.
Scan protocol Slides were scanned using an Axon 4100A Microarray Scanner (Axon Instruments) with GenePix Pro 5.0 software (Axon Instruments) and at a PMT count ratio (635/532) of about 1.
Description Chicken day six liver, Vehicle Control replicate 4
Data processing The results of image analysis were automatically merged with Excel files containing the clone identification number/plate address and gene name/function (from the highest BLAST score). Data in the VALUE column is based on the loess-normalized ratio of the loess-normalized log2 Alexa 555 and Alexa 647 values. The values are based on non-background corrected median values.
 
Submission date Jun 30, 2006
Last update date Aug 08, 2006
Contact name Larry Albert Cogburn
E-mail(s) cogburn@udel.edu
Phone 302-831-1335
Organization name University of Delaware
Department Animal and Food Sciences
Lab Avian Functional Genomics
Street address 531 South College Ave.
City Newark
State/province DE
ZIP/Postal code 19717
Country USA
 
Platform ID GPL1731
Series (1)
GSE5205 Analysis of Hepatic Gene Expression in Chickens with Hormonally-Induced Lean and Fat Phenotypes

Data table header descriptions
ID_REF
VALUE log2 of PRE_VALUE
lCy3_adj Loess-normalized log of Alexa 555 value
lCy5_adj Loess-normalized log of Alexa 647 value
F635 Median Alexa 647 raw intensity
B635 Median background value for Alexa 647
F532 Median Alexa 555 raw intensity
B532 Median background value for Alexa 555
Flags Flagged unsatisfactory values indicated by -50
UNF_VALUE Loess-normalized ratio
PRE_VALUE same as UNF_VALUE but with flagged values removed

Data table
ID_REF VALUE lCy3_adj lCy5_adj F635 Median B635 Median F532 Median B532 Median Flags UNF_VALUE PRE_VALUE
1.1.1 0.3188 7.412757295 7.093922195 113 67 206 134 0 1.247322995 1.247322995
1.1.2 7.332146895 6.882172226 96 67 198 128 -50 1.366016273
1.1.3 -0.0285 12.02492318 12.05344062 4377 71 4047 131 0 0.980427301 0.980427301
1.1.4 -0.2172 10.09977321 10.31695011 1252 69 1118 116 0 0.860247137 0.860247137
1.1.5 0.0503 7.659555463 7.609278906 168 67 235 113 0 1.035463397 1.035463397
1.1.6 0.1510 7.129004804 6.978048766 102 70 173 118 0 1.110305 1.110305
1.1.7 0.0202 7.219450097 7.199259027 121 69 181 113 0 1.014093777 1.014093777
1.1.8 -0.3318 10.40443717 10.73622287 1690 67 1368 110 0 0.794552416 0.794552416
1.1.9 -0.0390 11.24191119 11.28087194 2514 66 2397 106 0 0.97335586 0.97335586
1.1.10 -0.0719 8.981251763 9.053106605 499 63 538 104 0 0.951413999 0.951413999
1.1.11 6.539170298 6.375588541 66 63 117 104 -50 1.120064455
1.1.12 -0.2113 9.616049195 9.827363852 878 64 812 107 0 0.86374978 0.86374978
1.1.13 0.0193 13.39616916 13.37691369 11040 68 10388 109 0 1.013436345 1.013436345
1.1.14 -0.1678 11.83162883 11.9993946 4206 66 3548 110 0 0.890220255 0.890220255
1.1.15 -0.1978 11.84009587 12.03788298 4322 66 3567 109 0 0.871886889 0.871886889
1.1.16 -0.1310 7.436146192 7.567196028 161 64 204 108 0 0.913166705 0.913166705
1.1.17 -0.0299 12.45724028 12.487107 5950 65 5426 109 0 0.979510786 0.979510786
1.1.18 -0.0832 9.3046377 9.387814345 638 67 664 111 0 0.943976831 0.943976831
1.1.19 -0.1357 7.653624482 7.789319014 192 67 232 112 0 0.910231531 0.910231531
1.1.20 -0.1279 7.974136182 8.10205311 245 73 282 116 0 0.915151862 0.915151862

Total number of rows: 19200

Table truncated, full table size 1401 Kbytes.




Supplementary file Size Download File type/resource
GSM117520.tif.gz 32.1 Mb (ftp)(http) TIFF

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