|
| Status |
Public on Jun 12, 2009 |
| Title |
Test array design TA2_2520023_array 2_PMT gain 400 |
| Sample type |
RNA |
| |
|
| Source name |
Pooled RNA isolated from adult, juvenile and cocoon earthworms
|
| Organism |
Eisenia fetida |
| Characteristics |
tissue: whole body or cocoon
age: varied
|
| Treatment protocol |
Adults bearing clitelum, juveniles and cocoons were pulled out of the culture immediately before being used for RNA extraction.
|
| Growth protocol |
Earthworms were kept in moistened sphagnum peat (pH 6.5–7.5, moisture content 50%) and fed ad libitum on a diet of with Magic Worm Food (Carolina Biological Supply).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNeasy mini kit (QIAGEN, Valencia, CA) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the custom-designed Agilent test array for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on GenePix 4200AL scanner at PMT gain levels 400 and 500 using default setting (Scan resolution 5um, One channel-Green, Laser power 100%).
|
| Description |
Testing 244K 60-mer probes designed using eArray
|
| Data processing |
The scanned images were analyzed with GenePix 5 (Molecular Devices, Sunnyvale, CA) using default parameters and Agilient-provided GAL grid file 020023_D_20080421_gal to obtain signal intensities. The background-subtracted median signal intensity (SI) was reported. A threshold SI (mean + 2 × standard deviation of the lowest three spike-in RNA concentrations) was set for each array. Any spot with a SI smaller than the threshold was flagged as absent, whereas others were considered present and thus probes deposited on these spots were regarded as positive probes. Negative control spots were also checked to make sure their SI did not exceed those of the lowest spike-in RNAs.
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| |
|
| Submission date |
Jun 11, 2009 |
| Last update date |
Jun 11, 2009 |
| Contact name |
Ping Gong |
| E-mail(s) |
ping.gong@us.army.mil
|
| Phone |
(601) 634-3521
|
| Organization name |
US Army ERDC
|
| Department |
Environmental Laboratory
|
| Lab |
Environmental Genomics and Genetics
|
| Street address |
3909 Halls Ferry Road
|
| City |
Vicksburg |
| State/province |
MS |
| ZIP/Postal code |
39180 |
| Country |
USA |
| |
|
| Platform ID |
GPL8682 |
| Series (2) |
| GSE16548 |
Using pooled earthworm RNA to test 244K probes on TA-2 test array design 2520023 antisense 1 |
| GSE16551 |
Using pooled earthworm RNA to test 244K probes on TA-1, TA-2, TA-3, TA-4 test arrays |
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