|
| Status |
Public on Jun 01, 2011 |
| Title |
ME vs JE rep3 (Dye Swap) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
JE3
|
| Organism |
Pinus radiata |
| Characteristics |
development: juvenile
wood type: early
|
| Treatment protocol |
Developing xylem tissues from trees at juvneile (5 yrs) and mature (13 yrs) ages were collected on the same dates in spring (October) and autumn (March) to represent earlywood (EW) and latewood (LW) samples, respectively. To avoid the presence of compression wood in the samples, EW and LW tissues were collected from the same tree at breast height (1.3m) on opposite sides of the trunk perpendicular to the prevailing wind direction. The xylem tissues were scraped from the exposed xylem surface with a sharp chisel after removing the bark of the sampling area. Each time of sample collection was finished within two hours in the morning to minimize the fluctuated gene expression. The sampled tissues were immediately placed into liquid nitrogen in the field until stored in the lab at -80oC.
|
| Growth protocol |
Nine radiata pine trees (different genotypes) at juvenile (5 yrs) and mature (13 yrs) ages (based on wood ring number at breast height, 1.3m) each were sampled in commercial plantations at Bondo, NSW (35º 16' 44.04 S, 148º 26' 54.66 E). The 18 sampled trees were growing within 100 m of each other with similar field environment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Developing xylem tissues were ground and total RNA was extracted using the method of Chang et al (1993) with a slight modification
|
| Label |
Alexa Fluor 647
|
| Label protocol |
Equal amounts of total RNA (20 or 30 µg) of the two samples to be compared were reversely transcribed using Superscript reverse transcriptase, and generated fluorescently labelled cDNA using Alexa Fluor® 555-aha-dUTP and Alexa Fluor® 647-aha-dUTP, following the protocol of the SuperScriptTM Plus Direst cDNA Labelling System (Invitrogen, CA).
|
| |
|
| Channel 2 |
| Source name |
ME3
|
| Organism |
Pinus radiata |
| Characteristics |
development: mature
wood type: early
|
| Treatment protocol |
Developing xylem tissues from trees at juvneile (5 yrs) and mature (13 yrs) ages were collected on the same dates in spring (October) and autumn (March) to represent earlywood (EW) and latewood (LW) samples, respectively. To avoid the presence of compression wood in the samples, EW and LW tissues were collected from the same tree at breast height (1.3m) on opposite sides of the trunk perpendicular to the prevailing wind direction. The xylem tissues were scraped from the exposed xylem surface with a sharp chisel after removing the bark of the sampling area. Each time of sample collection was finished within two hours in the morning to minimize the fluctuated gene expression. The sampled tissues were immediately placed into liquid nitrogen in the field until stored in the lab at -80oC.
|
| Growth protocol |
Nine radiata pine trees (different genotypes) at juvenile (5 yrs) and mature (13 yrs) ages (based on wood ring number at breast height, 1.3m) each were sampled in commercial plantations at Bondo, NSW (35º 16' 44.04 S, 148º 26' 54.66 E). The 18 sampled trees were growing within 100 m of each other with similar field environment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Developing xylem tissues were ground and total RNA was extracted using the method of Chang et al (1993) with a slight modification
|
| Label |
Alexa Fluor 555
|
| Label protocol |
Equal amounts of total RNA (20 or 30 µg) of the two samples to be compared were reversely transcribed using Superscript reverse transcriptase, and generated fluorescently labelled cDNA using Alexa Fluor® 555-aha-dUTP and Alexa Fluor® 647-aha-dUTP, following the protocol of the SuperScriptTM Plus Direst cDNA Labelling System (Invitrogen, CA).
|
| |
|
| |
| Hybridization protocol |
Labelled cDNA was hybridised using the radiata pine 18K cDNA microarrays developed in CSIRO Plant Industry. Slides were placed in water bath for 16 h at 42 °C. Arrays then washed in 2x SSC (+0.1 % SDS) at 42 °C, 0.1x SSC (+0.1 % SDS), 0.1x SSC and 0.01x SSC at room temperature, each for 5 min prior to drying and scanning.
|
| Scan protocol |
Hybridized microarray slides were scanned using GenePix Personal 4100A microarray scanner (Axon Instruments, CA). Image pre-processing was performed using GenePix® Pro 4.0 and Acuity software (Axon Instruments, CA).
|
| Description |
Either JW or MW can be the experimental sample while the other as control sample.
|
| Data processing |
Data analysis was carried out using GEPAS v3.1. The mean values from each channel were log2 transformed and normalized using print-tip lowess and slide scale normalization.
|
| |
|
| Submission date |
Feb 25, 2010 |
| Last update date |
Jun 01, 2011 |
| Contact name |
Xinguo Li |
| E-mail(s) |
xinguo.li@csiro.au
|
| Phone |
61 2 6246 4848
|
| Fax |
61 2 6281 8312
|
| URL |
http://www.csiro.au
|
| Organization name |
CSIRO
|
| Department |
Plant Industry
|
| Lab |
Molecular Genetics
|
| Street address |
Building 602, Black Mountain Laboratories
|
| City |
Canberra |
| State/province |
ACT |
| ZIP/Postal code |
2601 |
| Country |
Australia |
| |
|
| Platform ID |
GPL8302 |
| Series (1) |
| GSE20535 |
Transcriptome reorganization during wood maturation in Pinus radiata |
|
