|
| Status |
Public on Jun 17, 2010 |
| Title |
daf16(M26); daf-2(e1370)_Heat Shocked_on Control RNAi_Fraction3_group2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Wild type (N2)_pooled mix
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
tissue: whole organism
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Each sample, including the reference pool, was subjected to two rounds of linear amplification using the MessageAmp kit from Ambion for the first round and the Amino Allyl Message Amplification II kit from Ambion for the second round. After amplification, each sample was assessed for yield and overall quality via Bioanlayzer (Agilent) and Nanodrop. Samples that did not pass QC were discarded, and not used in the final analysis. Post Labeling Reactive Dyes (Cy3 and Cy5) from Amersham were used in conjunction with the reagents and protocol from the Ambion Amino Allyl Message Amplification II Kit to label the aRNA. 5ug of aRNA plus aRNA spike were labeled (aRNA Spike: equal amounts of the 10 Stratagene Spot Repot Spikes were combined and then amplified 1 round, to make an aRNA Spot Report Spike mix). All controls were labeled with the Cy3 dye and all experimental samples were labeled with the Cy5 dye. The Cy3 and Cy5 sample pairs were combined and purified.
|
| |
|
| Channel 2 |
| Source name |
daf16(M26); daf-2(e1370) Heat Shocked on Control RNAi
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: daf16(M26); daf-2(e1370)
fraction: Fraction 3 - heavy polysomes
group: Group 2
treatment: Heat Shocked
tissue: whole organism
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Each sample, including the reference pool, was subjected to two rounds of linear amplification using the MessageAmp kit from Ambion for the first round and the Amino Allyl Message Amplification II kit from Ambion for the second round. After amplification, each sample was assessed for yield and overall quality via Bioanlayzer (Agilent) and Nanodrop. Samples that did not pass QC were discarded, and not used in the final analysis. Post Labeling Reactive Dyes (Cy3 and Cy5) from Amersham were used in conjunction with the reagents and protocol from the Ambion Amino Allyl Message Amplification II Kit to label the aRNA. 5ug of aRNA plus aRNA spike were labeled (aRNA Spike: equal amounts of the 10 Stratagene Spot Repot Spikes were combined and then amplified 1 round, to make an aRNA Spot Report Spike mix). All controls were labeled with the Cy3 dye and all experimental samples were labeled with the Cy5 dye. The Cy3 and Cy5 sample pairs were combined and purified.
|
| |
|
| |
| Hybridization protocol |
Hybridization was carried out using a Lucidea Slide Pro machine. All wash solutions were made using Ambion Pre-Made Solutions (Nuclease-free Water, 20% SDS and 20XSSC) and filtered sterilized with a 500ml, 0.22uM filter unit. All C. elegans arrays from the Washington University in St. Louis Genomic Sequencing Center arrive pretreated; no blocking step was necessary. To prepare the aRNA sample for hybridization, the labeled aRNA sample was brought up to a volume with Nuclease-free water and Dextran Sulfate/ Hybe Buffer mix was added to the aRNA sample. The mixture was warmed to 50oC. Using a Hamilton syringe the labeled sample was loaded into the Lucidea chamber. The hybridization program was run for 16hrs at 42oC with a cycling speed of 10µl/sec every 30 minutes. The array was then washed at a speed of 50ul/sec with the following wash buffers in succession: 2X SSC +0.1% SDS (warmed to 42oC for 1st wash solution), 2X SSC, 0.2XSSC, and a 95% ethanol drying step.
|
| Scan protocol |
The array slide was loaded into a Perkin Elmer Scan Array Express 2-Laser Scanner. A QuickScan was performed, which scans the first 3-4 rows of the array. A surface calibration was then performed using the Spot Report control spots on the array. After surface calibration, the PMT and POW settings were adjusted to have the control spots match in intensity. The array was then scanned. Images were quantitated using GenePix 5.0 software package which overlays the GAL file (provided by the Washington University in St. Louis Genomic Sequencing Center) on the two array TIFF images (Cy3 and Cy5). Pixels per spot, per channel were counted by the software and a gpr file was generated.
|
| Description |
The control RNAi is ‘empty’ vector L4440 RNAi feeding vector plasmid (1999 Firelab vector kit) transformed HT115(DE3). The empty vector RNAi was used as the food source for our array experiment.
|
| Data processing |
Arrays were normalized using LIMMA (Smyth & Speed 2003)
|
| |
|
| Submission date |
Jun 16, 2010 |
| Last update date |
Jun 16, 2010 |
| Contact name |
Serban Ciotlos |
| E-mail(s) |
smelov@buckinstitute.org
|
| Phone |
4085068553
|
| Organization name |
Buck Institute for Research on Aging
|
| Lab |
Melov Lab
|
| Street address |
8001 Redwood Blvd
|
| City |
Novato |
| State/province |
CA |
| ZIP/Postal code |
94945 |
| Country |
USA |
| |
|
| Platform ID |
GPL9458 |
| Series (1) |
| GSE22383 |
Insulin-like Signaling Determines Survival During Stress via Post Transcriptional Mechanisms in C. elegans. |
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