|
| Status |
Public on Mar 01, 2008 |
| Title |
maize_root_region3_ww-region2_ws_rep4-slideB |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
maize root region3 control 48h rep 4
|
| Organism |
Zea mays |
| Characteristics |
Region 3 of maize (Zea mays L. cv FR697) seedlings harvested 48h after transplant to vermiculite of high water potential.
|
| Treatment protocol |
See the growth protocol.
|
| Growth protocol |
Seeds were imbibed for 24 h in 1 mM CaSO4, then germinated for 28 h in vermiculite well-moistened with 1 mM CaSO4 at 29°C in the dark. Seedlings with primary roots 12-20 mm in length were transplanted into vermiculite mixed with pre-determined amounts of 1 mM CaSO4 to create either high (-0.03 MPa) or low (-1.6 MPa) water potential and grown under near-saturating humidity conditions to prevent further drying of the media. Root tips were collected at 48h after transplant to vermiculite of either high or low water potential.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The apical 12 mm of each root was sectioned into three regions based on previously-characterized longitudinal expansion rate profiles: region 1, 0-3 mm plus the root cap; region 2, 3-7 mm; region 3, 7-12 mm. Samples were immediately frozen in liquid nitrogen. Total RNA was isolated using Trizol reagent following the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA). Residual DNA was removed by Dnase I (Invitrogen, Carlsbad, CA) treatment for 15 min at room temperature, followed by use of RNeasy columns (Qiagen, Valencia, CA).
|
| Label |
Cy5
|
| Label protocol |
First strand cDNAs were synthesized from 50 μg of total RNA using anchored oligo(dT)24 primers with SuperScript III RT (Invitrogen, Carlsbad, CA), and aminoallyl-dUTP was incorporated into the cDNAs. The RNA template was removed by treatment with RnaseH (Invitrogen, Carlsbad, CA), and cDNAs were purified to remove unincorporated aminoallyl-dUTP using Microcon 30 spin concentrators (Millipore Corp., Bedford, MA). Following purification, monoreactive-Cye5 or Cye3 dyes (Amersham Biosciences Corp., Piscataway, NJ) were conjugated to aminoallyl-dUTP on the cDNAs and the unconjugated dye was removed using Qiagen PCR purification columns.
|
| |
|
| Channel 2 |
| Source name |
maize root region2 water stress 48h rep 4
|
| Organism |
Zea mays |
| Characteristics |
Region 2 of maize (Zea mays L. cv FR697) seedlings harvested 48h after transplant to vermiculite of low water potential.
|
| Treatment protocol |
See the growth protocol.
|
| Growth protocol |
Seeds were imbibed for 24 h in 1 mM CaSO4, then germinated for 28 h in vermiculite well-moistened with 1 mM CaSO4 at 29°C in the dark. Seedlings with primary roots 12-20 mm in length were transplanted into vermiculite mixed with pre-determined amounts of 1 mM CaSO4 to create either high (-0.03 MPa) or low (-1.6 MPa) water potential and grown under near-saturating humidity conditions to prevent further drying of the media. Root tips were collected at 48h after transplant to vermiculite of either high or low water potential.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The apical 12 mm of each root was sectioned into three regions based on previously-characterized longitudinal expansion rate profiles: region 1, 0-3 mm plus the root cap; region 2, 3-7 mm; region 3, 7-12 mm. Samples were immediately frozen in liquid nitrogen. Total RNA was isolated using Trizol reagent following the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA). Residual DNA was removed by Dnase I (Invitrogen, Carlsbad, CA) treatment for 15 min at room temperature, followed by use of RNeasy columns (Qiagen, Valencia, CA).
|
| Label |
Cy3
|
| Label protocol |
First strand cDNAs were synthesized from 50 μg of total RNA using anchored oligo(dT)24 primers with SuperScript III RT (Invitrogen, Carlsbad, CA), and aminoallyl-dUTP was incorporated into the cDNAs. The RNA template was removed by treatment with RnaseH (Invitrogen, Carlsbad, CA), and cDNAs were purified to remove unincorporated aminoallyl-dUTP using Microcon 30 spin concentrators (Millipore Corp., Bedford, MA). Following purification, monoreactive-Cye5 or Cye3 dyes (Amersham Biosciences Corp., Piscataway, NJ) were conjugated to aminoallyl-dUTP on the cDNAs and the unconjugated dye was removed using Qiagen PCR purification columns.
|
| |
|
| |
| Hybridization protocol |
The purified Cy3 and Cy5-labeled cDNAs were concentrated to 60 μl and hybridized to the maize oligonucleotide array (GPL1993) for 16-18 h at 42 ºC. Following hybridization, the arrays were washed three times, twice with medium stringency buffer (1X SSC, 0.2% SDS) and once with high stringency buffer (0.1X SSC, 0.2% SDS).
|
| Scan protocol |
Washed slides were dried and scanned immediately using a GenePix scanner (GenePix® 4000B, Axon Instruments, Inc.) at 532 nm (17 mW) and 635 nm (10 mW). GenePix Pro 4.1 software was then used to extract spot intensity data.
|
| Description |
This is the 4th of 4 biological replications, and one half of a dye-swap pair. Stressed region 2 was compared with control region 3 as a developmental control to the comparison of stressed region 2 with control region 2.
|
| Data processing |
Mean signal intensities were used without background correction and data from all 8 chips (4 reps with dye-swap) were normalized using the Loess transformation.
|
| |
|
| Submission date |
Oct 18, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
William G. Spollen |
| E-mail(s) |
spollenw@missouri.edu
|
| Phone |
573-884-8151
|
| Organization name |
University of Missouri-Columbia
|
| Department |
Informatics Research Core Facility
|
| Lab |
Scott A. Givan
|
| Street address |
113 Bond Life Sciences Center
|
| City |
Columbia |
| State/province |
MO |
| ZIP/Postal code |
65211 |
| Country |
USA |
| |
|
| Platform ID |
GPL1993 |
| Series (2) |
| GSE9369 |
Gene expression in Maize root region 2 at low water potential-developmental control |
| GSE9379 |
Gene expression in Maize root |
|
