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Sample GSM117555 Query DataSets for GSM117555
Status Public on Aug 09, 2006
Title Chicken day six liver, Thyroid Hormone 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, Thyroid Hormone 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 0.0142 7.002799377 6.988633893 89 59 183 120 0 1.009867128 1.009867128
1.1.2 -0.0613 7.019667479 7.08099486 96 57 183 115 0 0.958381934 0.958381934
1.1.3 0.0163 12.73725221 12.7209237 7138 54 6458 113 0 1.011382354 1.011382354
1.1.4 -0.1276 10.65916509 10.78671766 1830 54 1561 113 0 0.915383019 0.915383019
1.1.5 -0.1738 7.92467734 8.098492229 237 54 281 109 0 0.886495437 0.886495437
1.1.6 -0.1221 6.860847349 6.982975583 87 54 169 110 0 0.91883121 0.91883121
1.1.7 -0.0613 7.019667479 7.08099486 96 57 183 111 0 0.958381934 0.958381934
1.1.8 -0.0839 11.46767617 11.55157078 3137 58 2710 114 0 0.943507169 0.943507169
1.1.9 -0.0024 12.37648974 12.37890521 5606 57 5052 114 0 0.998327122 0.998327122
1.1.10 -0.4776 8.475937553 8.95358361 467 57 378 116 0 0.71814842 0.71814842
1.1.11 6.570522972 6.441403094 59 57 140 120 -50 1.093626326
1.1.12 -0.0743 10.63668376 10.71098194 1734 62 1539 124 0 0.949804061 0.949804061
1.1.13 0.1019 13.92863172 13.82668889 16034 66 14130 126 0 1.073217759 1.073217759
1.1.14 -0.0597 11.95455795 12.01428529 4339 68 3784 136 0 0.959445431 0.959445431
1.1.15 -0.0979 12.0736364 12.17151325 4844 67 4105 140 0 0.934407105 0.934407105
1.1.16 0.0292 7.457750563 7.42851759 134 73 226 139 0 1.020469435 1.020469435
1.1.17 -0.0752 12.2271727 12.30241322 5311 77 4560 136 0 0.949183861 0.949183861
1.1.18 -0.0678 9.77916063 9.84700469 928 74 872 131 0 0.954062669 0.954062669
1.1.19 -0.1149 7.59341941 7.708326835 169 69 239 133 0 0.923441564 0.923441564
1.1.20 -0.1388 8.439520755 8.578331577 352 77 377 140 0 0.90826751 0.90826751

Total number of rows: 19200

Table truncated, full table size 1390 Kbytes.




Supplementary file Size Download File type/resource
GSM117555.tif.gz 32.1 Mb (ftp)(http) TIFF

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