|
| Status |
Public on Sep 12, 2008 |
| Title |
+D7Smed_a |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Intact planaria 7 days after 100Gy (+D7Smed_a)
|
| Organism |
Schmidtea mediterranea |
| Characteristics |
Pool of 10 planarians 7 days after exposure to 100Gy
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent (Invitrogen), DNAse I treated (Qiagen) at room temperature for 15min, and cleaned up to remove any remaining organics or contaminants using Qiagen RNAeasy spin columns. RNA quality was assessed using an Agilent Bioanalyzer and amplified using the Ambion MessageAmp RNA amplication kit. The quality of the resulting amplified RNA (aRNA) was examined using the Agilent Bioanalyzer and quantified using a Nanodrop spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
1ug of aRNA was used as a template and random nonamers were used to prime first strand cDNA synthesis using Superscript II in the presence of Cy3-dCTP. The resulting cDNA/RNA hybrid was treated with 0.25 M NaOH to hydrolyze RNA. The first strand cDNA molecule was then purified using a Qiagen QIAquick PCR purification kit to remove unincorporated fluorescent nucleotides and primers. The purified labeled sample was subsequently concentrated through speedvac centrifugation and resuspended in Ambion SlideHyb Glass Array Hyb Buffer#1.
|
| |
|
| Channel 2 |
| Source name |
WTSmed_reference
|
| Organism |
Schmidtea mediterranea |
| Characteristics |
Pool of 10 intact wild type planarians
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent (Invitrogen), DNAse I treated (Qiagen) at room temperature for 15min, and cleaned up to remove any remaining organics or contaminants using Qiagen RNAeasy spin columns. RNA quality was assessed using an Agilent Bioanalyzer and amplified using the Ambion MessageAmp RNA amplication kit. The quality of the resulting amplified RNA (aRNA) was examined using the Agilent Bioanalyzer and quantified using a Nanodrop spectrophotometer.
|
| Label |
Cy5
|
| Label protocol |
1ug of aRNA was used as a template and random nonamers were used to prime first strand cDNA synthesis using Superscript II in the presence of Cy5-dCTP. The resulting cDNA/RNA hybrid was treated with 0.25 M NaOH to hydrolyze RNA. The first strand cDNA molecule was then purified using a Qiagen QIAquick PCR purification kit to remove unincorporated fluorescent nucleotides and primers. The purified labeled sample was subsequently concentrated through speedvac centrifugation and resuspended in Ambion SlideHyb Glass Array Hyb Buffer#1.
|
| |
|
| |
| Hybridization protocol |
The hybridization was carried out overnight at 42 degrees Celcius in a humid chamber
|
| Scan protocol |
The hybridized microarray slides were scanned using an Axon GenePix 4000B Scanner at a 10um/pixel resolution.
|
| Description |
Electronic spot quantification was accomplished using ImaGene software from Biodiscovery.
|
| Data processing |
The Cy3(exp)/Cy5(ref) ratio was calculated using Microsoft Excel, the data was mean scale normalized and log 2 transformed using Spotfire Decision Site Software for Functional Genomics (v7.0).
|
| |
|
| Submission date |
May 17, 2008 |
| Last update date |
Aug 17, 2008 |
| Contact name |
Alejandro Sánchez Alvarado |
| E-mail(s) |
sanchez@neuro.utah.edu
|
| URL |
http://planaria.neuro.utah.edu
|
| Organization name |
University of Utah School of Medicine
|
| Department |
Neurobiology and Anatomy
|
| Street address |
20N 1900E
|
| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84132-3401 |
| Country |
USA |
| |
|
| Platform ID |
GPL6871 |
| Series (1) |
| GSE11503 |
Expression Profiles of Planarain Stem Cells and their Immediate Division Progeny |
|
