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Sample GSM544555 Query DataSets for GSM544555
Status Public on Apr 19, 2011
Title stage_10_Cy3_143
Sample type RNA
 
Source name stage 10 embryo
Organism Fundulus heteroclitus
Characteristics source: wild population
developmental stage: stage 10 embryo
Treatment protocol Pools of frozen embryos collected at each developmental stage were used for RNA isolation, labeling, and microarray hybridization. Four pools of 25 embryos were used for stages 1-10, four pools of 15 embryos were used for stages 11-15, and 4 pools of 10 embryos were used for stages 16-40.
Growth protocol Eggs from multiple females were fertilized by sperm from multiple males, and excess sperm were removed. Fertilized embryos were maintained in Petri dishes half submerged in 15 ppt filtered seawater in a 25°C environmental chamber under light during the first two stages of development (2-cell stage). Embryos that successfully reached the 2-cell stage within 3 hours were incubated under a 16 hour light: 8 hour dark photoperiod at 25°C in the environmental chamber (818 Low Temperature Illuminated Incubator, Precision Scientific, USA).
Extracted molecule total RNA
Extraction protocol Embryo RNA was extracted using a Trizol buffer (Invitrogen, Carlsbad, CA, USA) followed by purification using the Qiagen RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA).
Label Cy3
Label protocol RNA for hybridization was prepared by one round of amplification (aRNA) using the Amino Allyl MessageAmp aRNA Kit (Ambion, Austin, TX, USA) to form copy template RNA by T7 amplification. Amino-allyl UTP was incorporated into targets during T7 transcription, and resulting amino-allyl aRNA was coupled to Cy3 and Cy5 dyes (GE Healthcare, Piscataway, NJ, USA).
 
Hybridization protocol Labeled aRNA samples (2 pmol dye/ul) were hybridized to slides in 10 ul of hybridization buffer [50% formamide buffer, 5x SSPE, 1% sodium dodecyl sulfate, 0.2 mg/ml bovine serum albumin, 1 mg/ml denatured salmon sperm DNA (Sigma), and 1 mg/ml RNAse free poly(A) RNA (Sigma)] for 44 hours at 42º C.
Scan protocol Arrays were scanned using a ScanArray Express 4000 (Perkin Elmer). Resulting 16 bit Tiff Images were quantified using ImaGene® (Biodiscovery, Inc.) spotfinding software.
Data processing Log2 measures of gene expression were normalized using a linear mixed model in JMP Genomics 3.2 (SAS, Cary, NC, USA) to remove the effects of dye (fixed effect) and array (random effect) following a joint regional and spatial Lowess transformation in MAANOVA version 0.98.8 for R to account for both intensity and spatial bias. The model was of the form yij = μ + Ai + Dj + (AxD)ij + εij where, yij is the signal from the ith array with dye j, μ is the sample mean, Ai and Dj are the overall variation in arrays (arrays 1-80) and dyes (Cy3 and Cy5), (AxD)ij is the array x dye interaction and εij is the stochastic error.
 
Submission date May 18, 2010
Last update date Apr 19, 2011
Contact name Marjorie F. Oleksiak
E-mail(s) moleksiak@rsmas.miami.edu
Organization name University of Miami
Department Marine Biology and Fisheries
Street address 4600 Rickenbacker Causeway
City Miami
State/province FL
ZIP/Postal code 33149
Country USA
 
Platform ID GPL10430
Series (1)
GSE21372 Gene expression throughout vertebrate embryogenesis

Data table header descriptions
ID_REF
VALUE Least square mean values from the mixed model normalization of log2, loess-transformed raw data

Data table
ID_REF VALUE
4 0.353206
5 0.173206
6 0.023206
7 2.033206
8 0.013206
9 0.333206
10 0.143206
11 0.243206
12 1.103206
13 0.133206
14 0.783206
15 0.183206
16 -0.09679
17 0.083206
18 0.183206
19 -0.27679
20 0.123206
21 -0.93679
22 -0.25679
23 0.023206

Total number of rows: 6857

Table truncated, full table size 92 Kbytes.




Supplementary file Size Download File type/resource
GSM544555.txt.gz 264.7 Kb (ftp)(http) TXT
Processed data included within Sample table

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