|
| Status |
Public on Mar 08, 2007 |
| Title |
Sh-Mt-Gi vs Mt-shoot, rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Mt-shoot, Cy3
|
| Organism |
Medicago truncatula |
| Characteristics |
Shoot tissues from Glomus intraradices mock-inoculated Medicago truncatula plants
|
| Treatment protocol |
2-week old M. truncatula seedlings were transplanted to 11-cm pot containing sterile Turface (eight plants per pot) and were mock-inoculated with the final distilled-water wash from the spore purification procedure. The plants were fertilized with half-strength Hoagland’s solution containing 0.02mM KH2PO4 twice weekly and harvested at 30 days post inoculation (dpi).
|
| Growth protocol |
Medicago truncatula cv Jemalong, line A17 plants were grown in growth rooms under a 16-h-light (25 °C ) / 8-h-dark (22 °C ) regime.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with Trizol Reagent® (Invitrogen) with an extra phenol-chloroform purification step, treated with RNase-free DNase (Promega) at 37°C for 20 minutes, and then purified using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
25μg total RNA was used to generate fluorescently labeled cDNA probes using SuperScriptTM Indirect cDNA Labeling System (Invitrogen: catalog no. L1014-02).
|
| |
|
| Channel 2 |
| Source name |
Sh-Mt-Gi, Cy5
|
| Organism |
Medicago truncatula |
| Characteristics |
Shoot tissues from Glomus intraradices inoculated Medicago truncatula plants
|
| Treatment protocol |
2-week old M. truncatula seedlings were transplanted to 11-cm pot containing sterile Turface (eight plants per pot) and about 8,000 G. intraradices spores were evenly inoculated onto the root of the seedlings. The plants were fertilized with half-strength Hoagland’s solution containing 0.02mM KH2PO4 twice weekly and harvested at 30 days post inoculation (dpi).
|
| Growth protocol |
Medicago truncatula cv Jemalong, line A17 plants were grown in growth rooms under a 16-h-light (25 °C ) / 8-h-dark (22 °C ) regime.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with Trizol Reagent® (Invitrogen) with an extra phenol-chloroform purification step, treated with RNase-free DNase (Promega) at 37°C for 20 minutes, and then purified using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
25μg total RNA was used to generate fluorescently labeled cDNA probes using SuperScriptTM Indirect cDNA Labeling System (Invitrogen: catalog no. L1014-02).
|
| |
|
| |
| Hybridization protocol |
Equivalent quantity of Cy3 labeled cDNA and Cy5 labeled cDNA probes were combined and evaporated to dryness in a speedvac at darkness. The combined dried cDNA targets were then suspended in 58 μl of hybridization solution containing 46 μl of 1.25x formamide based HybIt2™ hybridization solution (Telechem international, USA: catalog no. HHS2), 1 μl Poly (dA) (Invitrogen: catalog no. POLYA.GF) and 11 μl of H2O. The re-suspended cDNA probes were then incubated at 95°C for 5min and applied to a pre-warmed (50°C) slide, and covered with a clean glass LifterSlip (Erie Scientific company, USA: catalog no. 22X50I-2-4711). The slides were then sealed in Corning hybridization chambers (Corning Inc., USA: catalog no. 2551) and maintained at 43°C H2O bath with gentle shaking at 40 rpm for 16-20 hrs in the dark.
|
| Scan protocol |
Slides were scanned using a two-channel confocal microarray scanner (ScanArray5000, GSI Lumonics, MA, USA) and the associated ScanArray software (version3.1, Packard BioChip Technologies, MA, USA).
|
| Description |
Gene expression data of Sh-Mt-Gi vs Mt shoot samples
|
| Data processing |
Normalization and analysis of microarray data were performed using Genespring? software (version 6.2, Silicon Genetics, CA, USA). For each experiment, data from six slides were imported into Genespring? software and values for all spots on the arrays were normalized with per spot and per chip intensity dependent (Lowess) normalization, using 20% of the data to calculate the Lowess fit at each point.
|
| |
|
| Submission date |
Aug 07, 2006 |
| Last update date |
Sep 08, 2006 |
| Contact name |
Maria J. Harrison |
| E-mail(s) |
[email protected]
|
| Phone |
(607) 254-6472
|
| Fax |
(607) 254-6779
|
| Organization name |
Boyce Thompson Institute for Plant Research
|
| Lab |
Harrison
|
| Street address |
Tower Road, Cornell Campus
|
| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14853 |
| Country |
USA |
| |
|
| Platform ID |
GPL4059 |
| Series (2) |
| GSE5472 |
Gene expression data from Sh-Mt-Gi vs Mt shoot tissues |
| GSE5477 |
Arbuscular mycorrhizal symbioses |
|
