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Sample GSM34205 Query DataSets for GSM34205
Status Public on Oct 29, 2004
Title Triple-selected poly(A)+ human liver tissue RNA pooled from several individuals (HS_52361)
Sample type RNA
 
Source name Alexa555-labeled cDNA derived from human liver poly(A)+ RNA
Organism Homo sapiens
Extracted molecule total RNA
 
Description Triple-selected poly(A)+ human liver tissue RNA pooled from several individuals was obtained from Ambion (Austin, TX). First-strand cDNA was generated from 4 µg RNA/rxn with (RNase H-) M-MLV reverse transcriptase, primed with equimolar concentrations of oligo(dT18) and random decamers. The reactions were carried out at 42°C for 2 hours in the presence of 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.15 mM dTTP and 0.15 mM amino-allyl dUTP to facilitate the secondary labeling of an amine-reactive fluorescent conjugate.
Following reverse transcription the products were heated at 95°C for 5 minutes to denature the RNA:DNA hybrids and heat-inactivate the reverse transcriptase, after which the RNA template was hydrolyzed by incubating in 0.2 M NaOH at 65°C for 15 minutes. The reaction was then neutralized in 0.3 M HEPES (pH 7.0). Reverse transcription products from 20 separate reactions were produced in this manner and pooled to reduce technical variability between samples.
The cDNAs were precipitated in ethanol:isopropanol (1:1 v/v) and resuspended in 0.1 M NaHCO3 to facilitate coupling of Alexa Fluor 555 NHS esters (Molecular Probes, Eugene, OR) to the reactive groups of the amino-allyl dUTPs. Following incubation at room temperature for 1 hour, the labeled cDNAs were purified with CyScribe GFX glass fiber spin columns (Amersham Bioscience, Piscataway, NJ) and isopropanol-precipitated.
Microarrays were hybridized with 2-3 µg labeled cDNA in 320 µL hybridization buffer (50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20) for 20 hours at 50°C. Hybridizations were performed in disposable adhesive chambers (Grace BioLabs, Bend, OR) in a hybridization oven with constant agitation.
After hybridization, the arrays were washed on an orbital platform in non-stringent buffer (6× SSPE, 0.01% [v/v] Tween-20) for 10 minutes at room temperature, then in stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween-20) for 30 minutes at 45°C. This was followed by a 5-minute wash in non-stringent buffer and a 4-minute wash in 0.2X SSC.
The arrays were dried with compressed nitrogen. Fluorescence micrographs were acquired with an Axon 4000B laser scanner at 5 µm resolution and intensity data were extracted with NimbleScan software (NimbleGen Systems).
Keywords = liver tissue, poly(A)+ RNA
 
Submission date Oct 29, 2004
Last update date Oct 28, 2005
Contact name Paul Bertone
E-mail(s) paul.bertone@yale.edu
Phone (203) 432-5405
Organization name Yale University
Street address
City New Haven
State/province CT
ZIP/Postal code 06520
Country USA
 
Platform ID GPL1665
Series (1)
GSE1904 Maskless photolithography genome tiling experiment

Data table header descriptions
ID_REF
X X coordinate of the array feature
Y Y coordinate of the array feature
VALUE Median fluorescence signal intensity of the array feature, calculated over 9 pixels at 5 m resolution
SIGNAL_MEAN Mean fluorescence signal intensity of the array feature, calculated over 9 pixels at 5 m resolution
SIGNAL_STD Standard deviation of the fluorescence signal for the array feature

Data table
ID_REF X Y VALUE SIGNAL_MEAN SIGNAL_STD
1.1 1 1 177 182.88889 28.902615
1.3 1 3 97 96.22222 7.9179826
1.5 1 5 168 176.77777 40.877186
1.7 1 7 83 92.77778 19.626371
1.9 1 9 108.5 109.55556 22.978855
1.11 1 11 751.5 853.7778 194.84595
1.13 1 13 73 75.55556 11.577037
1.15 1 15 76 87.111115 20.593958
1.17 1 17 193.5 194 28.930952
1.19 1 19 92.5 97.333336 17.86057
1.21 1 21 95 98.55556 14.561173
1.23 1 23 64 77.44444 21.886703
1.25 1 25 196.5 200 28.350485
1.27 1 27 69.5 90 48.67751
1.29 1 29 62 70.333336 26.086395
1.31 1 31 146 157.44444 49.363728
1.33 1 33 176 177.22223 21.399637
1.35 1 35 76 91.44444 27.573135
1.37 1 37 206.5 214.55556 71.875084
1.39 1 39 55.5 56.77778 11.519307

Total number of rows: 389434

Table truncated, full table size 14590 Kbytes.




Supplementary data files not provided

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