|
| Status |
Public on Oct 26, 2017 |
| Title |
Pcan1H1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Traumatic xylem formed in wound margins
|
| Organism |
Pinus canariensis |
| Characteristics |
tissue: Traumatic xylem
Stage: H1
|
| Growth protocol |
Pines were grown in greenhouse, using 650 ml conical containers with 3:1 (v/v) peat:vermiculite. After the first year, trees were transferred to soil in experimental garden at UPM facilities, and grown under environmental conditions. At the moment of the beginning of this experiment, trees were 5 years old, approximately 2 m high and 7-10 cm diameter at the base.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from differentiating xylem samples stored at -80ºC, using the CTAB-LiCl precipitation method (Chang et al. 1993), and purified with the RNeasy Plant Mini Kit (Qiagen, CA, USA). Quantity of total RNA for each sample was measured with Nanodrop model ND-1000 (Thermo Scientific, MA, USA), and RNA quality was checked using the Experion Bioanalyzer (Bio-Rad, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
The Low Input Quick Amp Labeling Kit, Two-Color generates fluorescent cRNA (complimentary RNA) with 200 ng of total RNA for two-color processing. The method uses T7 RNA Polymerase Blend (red cap), which simultaneously amplifies target material and incorporates Cyanine 3-CTP or Cyanine 5-CTP.
|
| |
|
| Channel 2 |
| Source name |
Cambial cells differentiating to xylem in remote parts of the plant
|
| Organism |
Pinus canariensis |
| Characteristics |
tissue: Xylem (control)
Stage: H1
|
| Growth protocol |
Pines were grown in greenhouse, using 650 ml conical containers with 3:1 (v/v) peat:vermiculite. After the first year, trees were transferred to soil in experimental garden at UPM facilities, and grown under environmental conditions. At the moment of the beginning of this experiment, trees were 5 years old, approximately 2 m high and 7-10 cm diameter at the base.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from differentiating xylem samples stored at -80ºC, using the CTAB-LiCl precipitation method (Chang et al. 1993), and purified with the RNeasy Plant Mini Kit (Qiagen, CA, USA). Quantity of total RNA for each sample was measured with Nanodrop model ND-1000 (Thermo Scientific, MA, USA), and RNA quality was checked using the Experion Bioanalyzer (Bio-Rad, CA, USA).
|
| Label |
Cy5
|
| Label protocol |
The Low Input Quick Amp Labeling Kit, Two-Color generates fluorescent cRNA (complimentary RNA) with 200 ng of total RNA for two-color processing. The method uses T7 RNA Polymerase Blend (red cap), which simultaneously amplifies target material and incorporates Cyanine 3-CTP or Cyanine 5-CTP.
|
| |
|
| |
| Hybridization protocol |
Probes were mixed and hybridized to a 60K custom microarray (Agilent Technologies) for 17 hours at 65ºC in Gex Hybridization Buffer HI-RPM in a hybridization oven. Arrays were washed according to the manufacturer’s instructions, dried by centrifugation.
|
| Scan protocol |
Arrays were scanned at 3 mm resolution on Agilent DNA Microarrays Scanner (G2565BA, Agilent Technologies) and the images were analyzed with Feature Extraction software (Agilent Technologies).
|
| Description |
Pc13 CH0 (Cy3) vs Pc13 H0 (Cy5)
|
| Data processing |
For local background correction and normalization, the methods normexp and loess in LIMMA were used, respectively (Smyth and Speed 2003).
|
| |
|
| Submission date |
Aug 04, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Victor Chano |
| E-mail(s) |
vmchano@gmail.com
|
| Organization name |
Universidad Politecnica de Madrid
|
| Department |
Sistemas y Recursos Naturales
|
| Lab |
Genetica, Fisiologia e Historia Forestal
|
| Street address |
Ciudad Universitaria s/n
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28040 |
| Country |
Spain |
| |
|
| Platform ID |
GPL23849 |
| Series (1) |
|
