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Sample GSM34193 Query DataSets for GSM34193
Status Public on Oct 29, 2004
Title Triple-selected poly(A)+ human liver tissue RNA pooled from several individuals (HS_30213)
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 GPL1653
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 4942 5816 3190.195
1.3 1 3 2899 4416.222 4564.763
1.5 1 5 2608 3442.4443 1900.0045
1.7 1 7 1811.5 1891.6666 885.5069
1.9 1 9 3193.5 3620.7778 1482.2969
1.11 1 11 2860.5 5184 3838.2705
1.13 1 13 2372.5 2538.7778 402.87366
1.15 1 15 2577 3354.4443 1753.489
1.17 1 17 1125.5 1400.8889 736.25867
1.19 1 19 9384 10586.556 3042.0164
1.21 1 21 3042 3338 899.35114
1.23 1 23 3148.5 7178.6665 12057.294
1.25 1 25 2489 2865.889 1130.2811
1.27 1 27 2824 2931.5557 883.8152
1.29 1 29 2055.5 2500.5557 1092.3196
1.31 1 31 2714.5 3022.889 1552.7869
1.33 1 33 2922 3526.889 2297.4453
1.35 1 35 1876.5 1863.8889 270.75333
1.37 1 37 2934 5553.5557 4201.256
1.39 1 39 1706.5 1994.1111 778.48706

Total number of rows: 388996

Table truncated, full table size 15378 Kbytes.




Supplementary data files not provided

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