|
| Status |
Public on Aug 04, 2011 |
| Title |
M5-K6-DSRep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Tall Mokka shoot tip including first unopened leaf pair
|
| Organism |
Coffea arabica |
| Characteristics |
cultivar: Tall Mokka
|
| Treatment protocol |
Shoot tips including unopened leaves were collected from all trees.
|
| Growth protocol |
Tall Mokka and Typica trees were clonally propagated and maintained under similar environmental conditions in the fields at the Kunia experimental station of Hawaii Agriculture Research Center (HARC) on Oahu.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNeasy Plant mini kit following manufacturers instructions.
|
| Label |
Alexa 555
|
| Label protocol |
Total RNA was used to synthesize fluorescently labeled (Alexa Fluor 555 & Alexa Fluor 647) cDNA probes using the SuperScript Indirect cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA) following manufacturers instructions.
|
| |
|
| Channel 2 |
| Source name |
Typica shoot tip
|
| Organism |
Coffea arabica |
| Characteristics |
cultivar: Typica
|
| Treatment protocol |
Shoot tips including unopened leaves were collected from all trees.
|
| Growth protocol |
Tall Mokka and Typica trees were clonally propagated and maintained under similar environmental conditions in the fields at the Kunia experimental station of Hawaii Agriculture Research Center (HARC) on Oahu.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNeasy Plant mini kit following manufacturers instructions.
|
| Label |
Alexa 647
|
| Label protocol |
Total RNA was used to synthesize fluorescently labeled (Alexa Fluor 555 & Alexa Fluor 647) cDNA probes using the SuperScript Indirect cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA) following manufacturers instructions.
|
| |
|
| |
| Hybridization protocol |
Potato cDNA microarrays were prehybridized for 45 min in prehybridization buffer (5X SSC, 0.1% SDS, 1% BSA) preheated at 42°C. Prehybridized slides were washed twice in miliQ water for 5 min each, then washed for 2 min in isopropanol and air dried. Labelled cDNA probes were dissolved in 45 µl of 1X hybridization buffer (50% formamide, 5X SSC, and 0.1% SDS). 2 µl of EDTA (from 0.5M stock), 2 µl of polyA DNA (from 10 µg/ µl stock) and 0.75 µl of salmon sperm DNA (from 10 µg/µl stock) were added into the probe mix. Heat denatured probes were carefully applied to the microarray slides and incubated in hybridization chamber at 42°C for 16-20h. Hybridized slides were washed for 5 min in a low stringency wash buffer (2X SSC and 0.1% SDS) preheated to 42°C, followed by 10 min wash in a high-stringency wash buffer (0.05X SSC and 0.1% SDS) at room temperature. Slides were washed four times for one min each in 0.05X SSC, and dried using centrifuge.
|
| Scan protocol |
Scanned on GenePix Personal 4100A scanner (Molecular Devices Corporation, Sunnyvale, CA). Images were quantified using using GenePix Pro 6.0 (Molecular Devices Corporation, Sunnyvale, CA).
|
| Description |
65_MA5-KA6
Dye swap Replicate of 66_KA5-MA6, Total RNA from shoot tip including first unopened leaf of Tall Mokka and Typica.
|
| Data processing |
Per spot and per chip normalization based on the Lowess method was implemented using GeneSpring 7.0 (Agilent Technologies, Santa Clara, CA). Statistically significant differentially expressed genes were identified by applying one-way ANOVA analysis.
|
| |
|
| Submission date |
Aug 03, 2011 |
| Last update date |
Aug 04, 2011 |
| Contact name |
Ratnesh Singh |
| E-mail(s) |
ratnesh@hawaii.edu
|
| Phone |
808-621-1279
|
| Fax |
808-621-1399
|
| Organization name |
Hawaii Agriculture Research Center
|
| Street address |
94-340 Kunia Rd
|
| City |
Waipahu |
| State/province |
HI |
| ZIP/Postal code |
96797 |
| Country |
USA |
| |
|
| Platform ID |
GPL14110 |
| Series (1) |
| GSE31179 |
Shoot tip tissue: Tall Mokka vs Typica. |
|
