|
| Status |
Public on May 21, 2009 |
| Title |
LPS Vs. Normal (Sample 1 dye swap) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LPS
|
| Organism |
Holothuria glaberrima |
| Characteristics |
immuno-activated: Yes (LPS)
regeneration stage: n/a
|
| 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 |
Normal
|
| Organism |
Holothuria glaberrima |
| Characteristics |
regeneration stage: Normal
immuno-activated: No
|
| 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 |
|
