|
| Status |
Public on Dec 01, 2007 |
| Title |
Control N2 Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Amplified total RNA from untreated worms labeled with Cyanine-5 (red).
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
Wildtype (N2)
Sex: hermaphrodite
|
| Treatment protocol |
NGM 25oC 48h
|
| Growth protocol |
cultured from egg at 20oC prior to treatment. Shifted to 25oC for treatment for 48hr
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from pellets of 50 worms (each sample consisted of 50 picked worms stored at -80 in an eppendorf tube until extraction) using Stratagene's Absolute RNA Microprep kit (400805). Using the Microprep kit components and columns, the worm pellets were extracted using a BME and lysis buffer solution and washed with a column using salt and ethanol wash buffers (provided with the kit). RNA was then Dnased using the kit components and then washed with a salt and ethanol buffer and eluted in nuclease-free water. Sample concentration was assessed using a Nanodrop.The entire total RNA sample was then amplified using Ambion's Amino Allyl Message Amp II kit (AM1795 and AM1796). Following the protocol, the total RNA was amplified one round. The concentration was then assessed with a Nanodrop and the quality of the aRNA was verified using Agilent's Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
5ug's of aRNA, spiked with control aRNA (1-10 Spot Report Spikes), was dried with a speed vacuum in a 1.5ml eppendorf tube. Cy3/Cy5 CyDye Post Labeling Reactive Dyes from Amersham were re-suspended in DMSO (1 dye: 1 sample). The dried aRNA was re-suspended in coupling buffer, provided with the Ambion kit and mixed with the re-suspended dye. Controls were labeled with Cy3 and experimentals were labeled with Cy5. After the dyes coupled at room temperature they were purified using Ambion's provided columns and wash protocol. After elution, the labeled aRNA was fragmented using Ambion's Fragmentation kit (8740).
|
| |
|
| Channel 2 |
| Source name |
Total RNA from pooled mixed stage wildtype (N2) C. elegans, labeled with Cyanine-3 (green).
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
Wildtype (N2)
Sex: hermaphrodite
Mixed stage
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from pellets of 50 worms (each sample consisted of 50 picked worms stored at -80 in an eppendorf tube until extraction) using Stratagene's Absolute RNA Microprep kit (400805). Using the Microprep kit components and columns, the worm pellets were extracted using a BME and lysis buffer solution and washed with a column using salt and ethanol wash buffers (provided with the kit). RNA was then Dnased using the kit components and then washed with a salt and ethanol buffer and eluted in nuclease-free water. Sample concentration was assessed using a Nanodrop.The entire total RNA sample was then amplified using Ambion's Amino Allyl Message Amp II kit (AM1795 and AM1796). Following the protocol, the total RNA was amplified one round. The concentration was then assessed with a Nanodrop and the quality of the aRNA was verified using Agilent's Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
5ug's of aRNA, spiked with control aRNA (1-10 Spot Report Spikes), was dried with a speed vacuum in a 1.5ml eppendorf tube. Cy3/Cy5 CyDye Post Labeling Reactive Dyes from Amersham were re-suspended in DMSO (1 dye: 1 sample). The dried aRNA was re-suspended in coupling buffer, provided with the Ambion kit and mixed with the re-suspended dye. Controls were labeled with Cy3 and experimentals were labeled with Cy5. After the dyes coupled at room temperature they were purified using Ambion's provided columns and wash protocol. After elution, the labeled aRNA was fragmented using Ambion's Fragmentation kit (8740).
|
| |
|
| |
| Hybridization protocol |
Using a Lucidea Slide Pro machine, the labeled aRNA was hybridized to a c.elegan oligo array, purchased from Washington University. The arrays were treated prior to shipment, so after the Lucidea performed a standard chamber wash, the array was loaded (array side-up) into the chamber, which was then locked into place. The control and experimental aRNA samples (Cy3 and Cy5) were combined and brought up to 92ul's with nuc-free water. Then an equal amount of hybridization buffer consisting of Genisphere’s 2X Formamide hybe buffer (C600V600S25) and 50% Dextran sulfate (final concentration, 1X Hybridization buffer and 2.5% Dextran Sulfate) were added to the sample along with LNA blocker (CW3920) from Genisphere. This mixture was heated and using a syringe injected into the Lucidea slide chamber portal. The solution was circulated over night at 42 degrees Celsius at a 30 minute cycle 10ul/second. Then the Lucidea washed the array using 4 washes in succession (2XSSC + 0.1% SDS, 2XSSC, 0.2XSSC and isopropanol) and then dried the array with air.
|
| Scan protocol |
Each array was scanned at 10um, using a Scan Array Express scanner. Using the control spots on the array, the two channels, Cy3 and Cy5 were equilibrated during a pre-scan, and brought within an equivalent range (equal amounts of Spot Report aRNA were spiked into both the Cy3 and Cy5 samples prior to labeling). Once the PMT and POW settings were adjusted, the array was scanned. First scanning Cy5 then Cy3.
|
| Description |
Biological replicate 2 of 6. Wildtype(N2) untreated control.
|
| Data processing |
Each array was quantitated using Axon's GenePix 5.0 software and the provided gal file from Washington University. Both channels were loaded, inspected for defects and flagged accordingly. Then GenePix generated a gpr file to be used for downstream analysis.
|
| |
|
| Submission date |
Jun 07, 2007 |
| Last update date |
Jun 07, 2007 |
| Contact name |
Gawain McColl |
| E-mail(s) |
[email protected]
|
| Phone |
415 209 2093
|
| Fax |
412 209 2232
|
| Organization name |
Buck Institute for Age Research
|
| Street address |
8001 Redwood Blvd
|
| City |
Novato |
| State/province |
CA |
| ZIP/Postal code |
94945 |
| Country |
USA |
| |
|
| Platform ID |
GPL5367 |
| Series (1) |
| GSE8058 |
Wildtype (N2) C. elegans: Control vs. 10mM LiCl treated for 48hr at 25oC |
|
