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Sample GSM406328 Query DataSets for GSM406328
Status Public on May 21, 2009
Title 3 dpe vs. LPS (sample 7 dye swap)
Sample type RNA
 
Channel 1
Source name 3 dpe
Organism Holothuria glaberrima
Characteristics immuno-activated: No
regeneration stage: 3 dpe
Treatment protocol After n days (3, 7 or 14) of regeneration the tissues were colected and mRNA was extracted and labeled with Cy3 and Cy5 depending on the comparisons.
Growth protocol ESTs from regenerating and normal tissues were obtained from cDNA library construction from different stages of regeneration.
Extracted molecule total RNA
Extraction protocol Total RNA extracted using Trizol following manufacturer's instructions (Quiagen)
Label Cy3
Label protocol 1ug of polyA RNA was amplified using T7 promoter and reverse transcribed with 0.1M DTT, 10 mM dNTPmix, 5X 1st strand buffer, 50% PEG, inorganic phosphatase, Rnase Out and MMLV-RT enzimes. labeling was performed using T7 RNA polymerase and Cy3 and Cy5 CTP during 2 hours at 40º C.
After amplification-labeling reaction samples were purified with Qiagen's RNeasy spin columns. Amplified and labeled cRNAs were quantified. Samples over 150ng/ul amplification and dye incorporation larger than 13 were used for hybridization (much better yield than minimun recomended by Agilent)
 
Channel 2
Source name LPS
Organism Holothuria glaberrima
Characteristics regeneration stage: n/a
immuno-activated: Yes (LPS)
Treatment protocol After n days (3, 7 or 14) of regeneration the tissues were colected and mRNA was extracted and labeled with Cy3 and Cy5 depending on the comparisons.
Growth protocol ESTs from regenerating and normal tissues were obtained from cDNA library construction from different stages of regeneration.
Extracted molecule total RNA
Extraction protocol Total RNA extracted using Trizol following manufacturer's instructions (Quiagen)
Label Cy5
Label protocol 1ug of polyA RNA was amplified using T7 promoter and reverse transcribed with 0.1M DTT, 10 mM dNTPmix, 5X 1st strand buffer, 50% PEG, inorganic phosphatase, Rnase Out and MMLV-RT enzimes. labeling was performed using T7 RNA polymerase and Cy3 and Cy5 CTP during 2 hours at 40º C.
After amplification-labeling reaction samples were purified with Qiagen's RNeasy spin columns. Amplified and labeled cRNAs were quantified. Samples over 150ng/ul amplification and dye incorporation larger than 13 were used for hybridization (much better yield than minimun recomended by Agilent)
 
 
Hybridization protocol Samples were prepared as manufacturer's recommendations using the fragmentation step and hybridized at 65°C for 17hours in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequential
Scan protocol Slides were scaned on an Agilent G2505B Scanner. Resolution used =5um
Images were quantified using Agilent Feature Extraction Software (version A9.5.3.1)
Description Biological replicate with dye swap conditions
dpe: days post evisceration
Data processing Agilent Feature Extraction Software (v 9.5.3.1) was used for background substraction and LOWES normalization.
Statistical analyzes were also performed with two softwares: 1) using R (BioConductor package) using the original Feature extractor output files and 2) Using GenePix and Acuity (Axon Lab.) for differentially expressed genes and clustering (hierarchical and non hierarchical) analyzes
 
Submission date May 20, 2009
Last update date May 20, 2009
Contact name Pablo Andres Ortiz-Pineda
Organization name University of Massachusetts
Department Veterinary & Animal Sciences
Lab ISB 455
Street address 661 N Pleasant st
City Amherts
State/province MA
ZIP/Postal code 01002
Country USA
 
Platform ID GPL8574
Series (1)
GSE16182 Gene expression profile of regenerating intestine in the sea cucumber H. glaberrima

Data table header descriptions
ID_REF
VALUE normalized log10 ratio Cy5/Cy3

Data table
ID_REF VALUE
1 2.022215646e-001
2 0.000000000e+000
3 -1.026602066e-001
4 1.650195999e+000
5 6.438633834e-001
6 -3.880216357e-003
7 -2.138169765e-002
8 0.000000000e+000
9 -2.427013620e-002
10 0.000000000e+000
11 -2.052869067e-001
12 2.108913793e+000
13 -3.918257825e-001
14 0.000000000e+000
15 0.000000000e+000
16 9.306668303e-002
17 -9.166608593e-002
18 1.348402622e-002
19 4.616966009e-001
20 4.262228527e-001

Total number of rows: 15744

Table truncated, full table size 350 Kbytes.




Supplementary file Size Download File type/resource
GSM406328.txt.gz 4.5 Mb (ftp)(http) TXT
Processed data included within Sample table

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