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Sample GSM34080 Query DataSets for GSM34080
Status Public on Oct 29, 2004
Title Triple-selected poly(A)+ human liver tissue RNA pooled from several individuals (HS_11693)
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 GPL1545
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 71.5 79.333336 19.754745
1.3 1 3 67.5 70.55556 13.4639435
1.5 1 5 76.5 87.77778 27.49899
1.7 1 7 60 60.555557 9.029273
1.9 1 9 61 59.11111 12.333333
1.11 1 11 56.5 62.88889 13.457753
1.13 1 13 61.5 61 12.776933
1.15 1 15 61 65.888885 12.7421
1.17 1 17 72 76.55556 24.648079
1.19 1 19 136 141.55556 32.245586
1.21 1 21 62 68.888885 16.305248
1.23 1 23 75.5 71 18.814888
1.25 1 25 70 71.77778 17.788885
1.27 1 27 73 76.888885 11.87902
1.29 1 29 61 66.22222 16.19242
1.31 1 31 70.5 68.111115 13.128637
1.33 1 33 65 62.22222 13.065008
1.35 1 35 64.5 70.44444 14.379769
1.37 1 37 67 83.111115 33.46806
1.39 1 39 64 82.666664 44.62903

Total number of rows: 389434

Table truncated, full table size 14599 Kbytes.




Supplementary data files not provided

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