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Sample GSM34094 Query DataSets for GSM34094
Status Public on Oct 29, 2004
Title Triple-selected poly(A)+ human liver tissue RNA pooled from several individuals (HS_12863)
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 GPL1559
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 1 144.5 178.66667 65.84071
1.3 1 3 174.5 182.44444 36.03856
1.5 1 5 146 148.66667 47.966133
1.7 1 7 213.5 335.66666 265.06085
1.9 1 9 166 192.55556 55.686874
1.11 1 11 147 152.44444 34.464153
1.13 1 13 173 178.11111 43.472534
1.15 1 15 169.5 182.44444 64.0822
1.17 1 17 152 159.11111 41.97155
1.19 1 19 173 167.88889 31.95483
1.21 1 21 181.5 168 56.747246
1.23 1 23 176 188.66667 65.024994
1.25 1 25 171 169.33333 41.952354
1.27 1 27 179.5 189.55556 34.09953
1.29 1 29 178.5 210.11111 81.48381
1.31 1 31 134 147.88889 28.855001
1.33 1 33 162 170.66667 36.687874
1.35 1 35 193.5 203.33333 65.59726
1.37 1 37 160.5 199 80.39745
1.39 1 39 201 197.22223 52.22973

Total number of rows: 389434

Table truncated, full table size 14989 Kbytes.




Supplementary data files not provided

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