|
| Status |
Public on Oct 22, 2011 |
| Title |
Leaf-Line3-2mMthen20mM-11PMday1-rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Line3 grown with 2mM and then 20mM ammonium nitrate sampled at 11pm_day1
|
| Organism |
Zea mays |
| Characteristics |
genotype: Monsanto Z. mays line 3
developmental stage: V6
tissue: Leaf tissue from 4 plants
growth nutrient: 2mM and then 20mM NH4NO3
nitrogen condition: N02T20: Limiting Nitrogen Supplemented
sampletime: 11PM day1
|
| Treatment protocol |
The treatment is the growth condition (see growth protocol).
|
| Growth protocol |
The soil used for filling the pots is Hummert’s Metro Mix 200 (~120g/pot), without a starter fertilizer. The nutrients are dispensed in the form of full strength Hoagland nutrient solution containing precise amounts of N added (2mM NH4NO3 for low N screening and 20mM NH4NO3 for high N screening runs). The supplemented low N samples (N02T20) were dispensed a 20mM NH4NO3 solution 2 hours prior to first being sampled. Each pot is manually dispensed 100ml of nutrient solution three times a week on alternate days starting at 10 days after planting. The plants are harvested after 4 weeks for the low N treatment and 3 weeks for the sufficient N treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mature leaf tissues are grounded using pestle and mortar with liquid nitrogen. 150mg-250mg grounded tissues were aliquoted to each Lysing Matix E tube (Qbiogen). RNA extraction was done using modified CTAB (0.5% CTAB, 0.05M Tris-HCl, 0.8M Nacl, 10mM EDTA/PH8.0) method using Qbiogen Fast Prep instrument. 750 ul of CTAB solution and 400ul of Phenol (PH4, from Amersco) were added to ground tissue. Fasta Prep beat was performed for 45 sec at setting 6. Later contents were centrifuged at 10,000g/10min.Supernatant was extracted twice with Phenol:Chloroform:Isoamyl alcohol (24:24:1) mixture. RNA in the upper phase was precipitated with ethanol. Precipitated RNA pellet was suspended in RNase free water (85ul) and treated with 5ul DNase I (2u/ul, Ambion) for 30min at 37C. DNase treated RNA was purified using RNeasy pink column (Qiagen). Quality of the purified RNA was determined by using Beckman DU640 spectrophotometer and Agilent Bio-analyzer 2100. The A260/A280 O.D. ratio of RNA samples measured using Beckman DU640 spectrophotometer was between 1.8 and 2.3. The 28s/18s ratio determined by using Agilent Bioanalyzer was above 0.8.
|
| Label |
biotin
|
| Label protocol |
For labeling, 500ng of purified total RNA was used as input. TargetAmp™ 1-Round Biotin-aRNA Amplification Kit 105 kit from Epicentre (TAB1R80524) was used in amplification and labeling process following the kit protocol. In the end, Biotin-labelind-aRNA probe was generated and QCed by OD reading (yield) and Agilent bioanalyzer (quality).
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| |
|
| Hybridization protocol |
Affymetrix GeneChips, custom made for Monsanto Company, were allowed to equilibrate to RT and 1X hybridization buffer (0.10M MES, 0.96M NaCl, 0.02M EDTA and 0.01% Tween) was brought to 45C. Chips were loaded with 1X hybridization buffer and incubated at 45C for 10min with rotation of 60rpm in a Hybridization Oven 640 (Affymetrix). During this time samples were prepared as follows: 20X eukaryotic hybridization controls (Affymetrix) were heated at 65C for 5min. 7.5ug labeled DNA was brought to 100ul with molecular biology grade water and the following were added: 4.0ul of control oligo B2 (Affymetrix), 12ul 20X eukaryotic hybridization controls, 2.4ul 10mg/ml salmon sperm DNA (Invitrogen), 2.4ul 50mg/ml acetylated BSA (Invitrogen) and 120ul 2X hybridization buffer. Samples were then heated at 99C for 5min, followed by 45C for 5min. Samples were centrifuged at 14,000g for 5min at RT to pellet any particulate matter. During this centrifugation, chips were removed from the hybridization oven and drained of the 1X hybridization buffer; replacing it with the hybridization cocktail after centrifugation. Chips were then placed into the hybridization oven and incubated for 16h at 45C with a rotation of 60rpm.
|
| Scan protocol |
Hybridized GeneChips were washed, stained and scanned according to the manufacturer’s instructions using the GeneChip Fluidics Station 450 (Affymetrix), an Affymetrix GeneChip Scanner 3000 and the GeneChip Operating Software (GCOS) (Affymetrix).
|
| Description |
Line3_N02T20_D1_11pm_R3
|
| Data processing |
Data from Affymetrix GCOS system were normalized by RMA algorithm in Partek Genomics Suite software and then scaled to 75th percentile of 500 over all chips.
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| |
|
| Submission date |
Sep 24, 2011 |
| Last update date |
Oct 22, 2011 |
| Contact name |
Jim Morrell |
| E-mail(s) |
[email protected]
|
| Organization name |
Monsanto Co.
|
| Department |
Biotechnology
|
| Street address |
800 North Lindbergh Blvd
|
| City |
Saint Louis |
| State/province |
Missouri |
| ZIP/Postal code |
63167 |
| Country |
USA |
| |
|
| Platform ID |
GPL14616 |
| Series (1) |
| GSE32361 |
Gene expression biomarkers provide sensitive indicators of in planta nitrogen status in Maize |
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