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Sample GSM34073 Query DataSets for GSM34073
Status Public on Oct 29, 2004
Title Triple-selected poly(A)+ human liver tissue RNA pooled from several individuals (HS_11648)
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 GPL1539
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 84.5 82.333336 12.519984
1.3 1 3 65 67.44444 9.540848
1.5 1 5 82.5 88.22222 24.868544
1.7 1 7 66 66.666664 10.392304
1.9 1 9 90.5 100.111115 38.426697
1.11 1 11 69.5 71.333336 26.818836
1.13 1 13 81.5 97.888885 32.66284
1.15 1 15 55 62 19.54482
1.17 1 17 76.5 81.111115 26.511528
1.19 1 19 77 79.111115 16.78872
1.21 1 21 69.5 78.55556 21.714691
1.23 1 23 81.5 81.888885 23.261795
1.25 1 25 80 87.55556 27.336382
1.27 1 27 63.5 61.22222 15.393
1.29 1 29 64.5 67.333336 7.0356236
1.31 1 31 66 69.44444 13.333333
1.33 1 33 84.5 95 26.072016
1.35 1 35 85.5 84 15.182226
1.37 1 37 70.5 72.22222 14.575474
1.39 1 39 66 85 20.603397

Total number of rows: 389434

Table truncated, full table size 14627 Kbytes.




Supplementary data files not provided

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