|
| Status |
Public on May 31, 2007 |
| Title |
Control_vs_Cold_CT6748_2H_R2_T2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Rice (Oryza sativa) seedlings at 2 hours at control temperature (29C)
|
| Organism |
Oryza sativa |
| Characteristics |
Rice whole seedlings (coleoptile, radicle, prophyl), CT6748-8-CA-17, S3-stage.
|
| Biomaterial provider |
Benildo G. de los Reyes, Dept. of Biological Sciences, University of Maine
|
| Treatment protocol |
Control samples (seedlings at S3 stage) were incubated at ambient temperature (29C) for 2 hrs.
|
| Growth protocol |
Seedlings were germinated in ample water and allowed to develop to S3-stage at ambient temperature (29C) in a growth chamber.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from control seedlings using the TriZol extraction method according to manufacturer's (Invitrogen) protocol.
|
| Label |
Cy5
|
| Label protocol |
The target RNA sample was processed in a two-step procedure using the CyScribe post-cDNA labeling kit (Amersham-GE Healthcare). The cDNA synthesis reaction was primed with oligo-dT catalyzed by CyScript reverse transcriptase. Aminoallyl-dUTPs were incorporated into first strand cDNA with an optimized nucleotide mix supplied in the kit. In the second step, cDNA samples with aminoallyl modification were chemically labeled with Cy5 fluorescent dye.
|
| |
|
| Channel 2 |
| Source name |
Rice (Oryza sativa) seedlings at 2 hours under cold stress (10C)
|
| Organism |
Oryza sativa |
| Characteristics |
Rice whole seedlings (coleoptile, radicle, prophyl), CT6748-8-CA-17, S3-stage.
|
| Biomaterial provider |
Benildo G. de los Reyes, Dept. of Biological Sciences, University of Maine
|
| Treatment protocol |
Treatment samples (seedlings at S3 stage) were incubated at low temperature (10C) for 2 hrs
|
| Growth protocol |
Seedlings were germinated in ample water and allowed to develop to S3-stage at ambient temperature (29C) in a growth chamber.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from cold-treated seedlings using the TriZol extraction method according to manufacturer's (Invitrogen) protocol.
|
| Label |
Cy3
|
| Label protocol |
The target RNA sample was processed in a two-step procedure using the CyScribe post-cDNA labeling kit (Amersham-GE Healthcare). The cDNA synthesis reaction was primed with oligo-dT catalyzed by CyScript reverse transcriptase. Aminoallyl-dUTPs were incorporated into first strand cDNA with an optimized nucleotide mix supplied in the kit. In the second step, cDNA samples with aminoallyl modification were chemically labeled with Cy3 fluorescent dye.
|
| |
|
| |
| Hybridization protocol |
Equal amounts of Cy5- and Cy3-labeled samples were combined, dried by speed-vac centrifugation and then dissolved in 1x hybridization buffer (Amersham-GE Healthcare) with 50% formamide. Hybridization was performed by applying the labeled mixture of control and cold stress samples onto the area of the microarray slide corresponding to the spot matrix and then covered with cover slip (Corning Life Sciences). The hybridization assembly was mounted in a tightly sealed and humidified portable hybridization cassette (Telechem International). Hybridization cassetes were wrapped with aluminum foil and incubated in 42C water bath for 16 to 18 hours. Following hybridization, the cover slips were gently peeled-off and the slides were washed in three stringency buffer solutions (TeleChem International): Buffer 1, 2x SSC (3M sodium chloride, 0.3M sodium citrate) plus 1% sarcosyl; Buffer 2, 2x SSC; Buffer 3, 0.2x SSC. All washing steps were carried out at room temperature for 5 minutes with moderate agitation.
|
| Scan protocol |
Microarrays were scanned at 635nm and 532nm using the Axon 4000A scanner. Scan image was acquired and processed by the GenePix Pro 3.0.
|
| Description |
This experiment was performed with two independent biological replicates (R1 and R2). The specific hybridization experiment described here represents the second of the two technical replicates (T2) within biological replicate R2.
|
| Data processing |
Quality flagging was performed on the microarray scans using GenePix Pro 3.0. A complex quality control query was used to identify and select high quality spots (features) from the entire microarray matrix. Separate analysis was performed on the duplicate panels (tecnical replicate). This query was formulated to exclude features that belong to any of the following filter criteria: 1) The feature intensity is near background level; 2) The feature is irregular or not uniform (spotting irregularity); and 3) The background around the feature is not uniform (hybridization irregularity). Only the features that passed these stringent filter criteria were included in subsequent bioinformatic analysis of the gene expression data. Gene expression data in each image was normalized globally prior to high level analysis. VALUES represent the log of the ratio between the background subtracted intensity values at Cy5 (635 nm) and Cy3 (532 nm) (635/532). All high level analysis was performed by Acuity bionformatics Suite (Axon Instruments).
|
| |
|
| Submission date |
Feb 19, 2007 |
| Last update date |
Feb 27, 2007 |
| Contact name |
Benildo G de los Reyes |
| E-mail(s) |
benildo.de@maine.edu
|
| Phone |
207-581-2564
|
| Fax |
2-7-581-2537
|
| Organization name |
University of Maine
|
| Department |
School of Biology and Ecology
|
| Lab |
De los Reyes Lab
|
| Street address |
5735 Hitchner Hall
|
| City |
Orono |
| State/province |
ME |
| ZIP/Postal code |
04469 |
| Country |
USA |
| |
|
| Platform ID |
GPL4878 |
| Series (1) |
| GSE7071 |
Reactive oxygen species trigger a regulatory module involved in the early responses of rice seedlings to cold stress |
|
| Data table header descriptions |
| ID_REF |
|
| EST ID |
dBEST Acc |
| Block |
Block of Spotting in the slide |
| Row |
Row of the Block |
| Column |
Column of the Block |
| F635 Median |
Feature Median Intensity Control Channel F635 |
| F635 Mean |
Feature Mean Intensity Control Channel F635 |
| F635 SD |
Standard deviation of F635 |
| F532 Median |
Feature Median Intensity Test Channel F532 |
| F532 Mean |
Feature Mean Intensity Test Channel F532 |
| F532 SD |
Standard deviation of F532 |
| Ratio of Medians (635/532) |
|
| Ratio of Means (635/532) |
|
| Median of Ratios (635/532) |
|
| Mean of Ratios (635/532) |
|
| Ratios SD (635/532) |
|
| VALUE |
Log Ratio (635/532) |
| F635 Median - B635 |
Background Corrected Feature Median Intensity Control Channel F635 |
| F532 Median - B532 |
Background Corrected Feature Median Intensity Control Channel F532 |
| F635 Mean - B635 |
Background Corrected Feature Median Intensity Control Channel F635 |
| F532 Mean - B532 |
Background Corrected Feature Median Intensity Control Channel F532 |
