|
| Status |
Public on Jan 15, 2008 |
| Title |
WT Rice branching panicle vs. dsRNAiRFL |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT rice TP309 branching panicles
|
| Organism |
Oryza sativa |
| Characteristics |
Rice TP309; wild type branching panicles
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from WT rice panicles using Tri Reagent (Sigma) and purified by RNeasy cleanup kit (Qiagen). RNA profile and integrity of RNA was checked by Agilent’s Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
cDNA was synthesized from tortal RNA by RT-PCR and was used as templete for Cy3 labeled cRNA synthesis using T7 dependent in vitro transcription system.
|
| |
|
| Channel 2 |
| Source name |
Transgenic rice panicles knock down for RFL
|
| Organism |
Oryza sativa |
| Characteristics |
Transgenic rice branching panicles of similar developmental stage to WT where a transcription regulator, RFL has been knocked down by double stranded RNA interference
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from RFL knock down rice panicles using Tri Reagent (Sigma) and purified by RNeasy cleanup kit (Qiagen). RNA profile and integrity of RNA was checked by Agilent’s Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
cDNA was synthesized from tortal RNA by RT-PCR and was used as templete for Cy5 labeled cRNA synthesis using T7 dependent in vitro transcription system.
|
| |
|
| |
| Hybridization protocol |
Probe fragmentation: Fragmentation reaction was as-
cRNA Cy3 750 ng,cRNA Cy5 750 ng,Blocking agent 50 ul,Water variable, 25 X fragmentation buffer,10 ul to 250 ul total volume.
Incubate at 60C for 30 mins. Add 250 ul of 2X hybridization buffer.
Use 490 ul for each slide and hybridize at 65C for 17 hrs at 5 rpm speed.
|
| Scan protocol |
Washing was done according to Agilent’s instructions using wash buffer 1 followed by wash buffer 2. Slides were scanned using Agilent DNA Microarray Scanner (G2565BA).
|
| Description |
Image quality was checked with the help of-
1. Four green spots and each corner of the image
2. SpikeIn A and B control spots across the image
3. Evenness of the background in log-scale mode
|
| Data processing |
The TIFF image was used to extract raw and processed data using Agilent’s Feature Extraction software (FE v8.1). The normalization was done using GeneSpring GX version 7.2 using the recommended Per Spot and Per Chip: Intensity dependent (Lowess) normalization. Number of non-control spots with ratio 0.25 and below were considered as down regulated spots.
|
| |
|
| Submission date |
Jan 08, 2008 |
| Last update date |
Jan 22, 2008 |
| Contact name |
Nagashree N Rao |
| E-mail(s) |
nagashri@mcbl.iisc.ernet.in
|
| Phone |
91-80-22932681
|
| Fax |
91-80-23600168
|
| URL |
http://mcbl.iisc.ernet.in
|
| Organization name |
Indian Institute of Science
|
| Department |
Microbiology and Cell Biology
|
| Lab |
Prof. Usha Vijayraghavan
|
| Street address |
C.V. Raman Avenue
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560012 |
| Country |
India |
| |
|
| Platform ID |
GPL892 |
| Series (1) |
| GSE10098 |
Identification of downstream genes regulated by RFL |
|
