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Sample GSM242868 Query DataSets for GSM242868
Status Public on Nov 11, 2008
Title L1 vs L2_2M
Sample type RNA
 
Channel 1
Source name L2 of a SAM. L2 is a corpus, which will become internal part the matured plant.
Organism Zea mays
Characteristics Inbred line: B73
Developmental stage: 14-day old seedlings
Sample name: L2_2
Growth protocol Maize (Zea mays inbred line B73) kernels were planted ~2 cm deep in plastic pots (8.5 cm x 8.5 cm wide at the top and 7.5 cm deep) filled with SB 300 Universal (Sun Gro Horticulture). Pots were placed in an environmental control room (PGW-40, Percival Scientific). The light intensity at the growth medium surface was kept between 760 to 810 mol/m2s as measured with a quantum meter (Model QMSW, Apogee Instruments). Temperature and light cycles were set at 25C with 15-hour light conditions and at 20C with 9-hour dark conditions. Pots were watered with a solution containing 0.7 mM calcium nitrate. Tissue blocks including SAMs (3 mm x 2 mm x 1 mm) were harvested from 14-day-old seedlings and processed to prepare paraffin sections. This protocol is also available at our project web site:
http://maize-meristems.plantgenomics.iastate.edu/resources/protocols/
Extracted molecule total RNA
Extraction protocol PicoPure RNA Isolation Kit (Arcturus/Molecular Devices) was used to extract total RNA from LCM-collected L1 and L2 cells of maize SAMs according to the manual. The RNA samples were treated with RNase-free DNase I (Stratagene) on column using the DNase Incubation Buffer provided with PALM RNA ExtractionKit (P.A.L.M. Microlaser Technologies) according to the manual of PicoPure RNA Isolation Kit. Approximately 1 ng of Dnase I-treated total RNA from each sample was then amplified with RiboAmp HS RNA Amplification Kit (Arcturus/Molecular Devices).
Label Cy3
Label protocol Five to ten micrograms of amplified RNA (within each replication the same amount of amplified RNA was used for both L1 and L2) was labeled with Cy3 or Cy5 according to Nakazono et al. (2003, Plant Cell 15: 583-596) with slight modifications. Details of fluorescent target synthesis are available at our project website (http://maize-meristems.plantgenomics.iastate.edu/resources/protocols/). To remove dye-specific effects in the statistical analyses, Cy dyes were swapped between the RNA samples with odd and even numbers of the replications.
 
Channel 2
Source name L1 of a shoot apical meristem (SAM). L1 is a single cell layered tunica, which will become epidermal cell layers of the matured plant.
Organism Zea mays
Characteristics Inbred line: B73
Developmental stage: Vegetative SAMs from 14-day old seedlings
Sample name: L1_2
Growth protocol Maize (Zea mays inbred line B73) kernels were planted ~2 cm deep in plastic pots (8.5 cm x 8.5 cm wide at the top and 7.5 cm deep) filled with SB 300 Universal (Sun Gro Horticulture). Pots were placed in an environmental control room (PGW-40, Percival Scientific). The light intensity at the growth medium surface was kept between 760 to 810 mol/m2s as measured with a quantum meter (Model QMSW, Apogee Instruments). Temperature and light cycles were set at 25C with 15-hour light conditions and at 20C with 9-hour dark conditions. Pots were watered with a solution containing 0.7 mM calcium nitrate. Tissue blocks including SAMs (3 mm x 2 mm x 1 mm) were harvested from 14-day-old seedlings and processed to prepare paraffin sections. This protocol is also available at our project web site:
http://maize-meristems.plantgenomics.iastate.edu/resources/protocols/
Extracted molecule total RNA
Extraction protocol PicoPure RNA Isolation Kit (Arcturus/Molecular Devices) was used to extract total RNA from LCM-collected L1 and L2 cells of maize SAMs according to the manual. The RNA samples were treated with RNase-free DNase I (Stratagene) on column using the DNase Incubation Buffer provided with PALM RNA ExtractionKit (P.A.L.M. Microlaser Technologies) according to the manual of PicoPure RNA Isolation Kit. Approximately 1 ng of Dnase I-treated total RNA from each sample was then amplified with RiboAmp HS RNA Amplification Kit (Arcturus/Molecular Devices).
Label Cy5
Label protocol Five to ten micrograms of amplified RNA (within each replication the same amount of amplified RNA was used for both L1 and L2) was labeled with Cy3 or Cy5 according to Nakazono et al. (2003, Plant Cell 15: 583-596) with slight modifications. Details of fluorescent target synthesis are available at our project website (http://maize-meristems.plantgenomics.iastate.edu/resources/protocols/). To remove dye-specific effects in the statistical analyses, Cy dyes were swapped between the RNA samples with odd and even numbers of the replications.
 
 
Hybridization protocol Microarray slides were prehybridized in 5X SSC (1X SSC is 0.15 M NaCl and 0.015 M sodium citrate), 0.1% SDS, and 0.1 mg/ml bovine serum albumin at 42oC for 45 min, then rinsed with autoclaved, Millipore water, dipped in isopropanol, and dried by centrifugation. Each target was resuspended in 30 l hybridization solution (5X SSC, 0.1% SDS, 0.2 g/l of yeast tRNA, 0.2 g/l of polyadenylic acid, 25% super-pure (99.5%) formamide (Fisher Scientific). Targets hybridized to the same slide were mixed, heated at 95oC for 3 min, collected by centrifugation, applied to a prehybridized microarray slide and covered with a 24 x 60 mm LiferSlip (Erie Scientific Company). Ten microliters of 3X SSC were added to each of the two slots of the hybridization chamber (TeleChem). The array was then sealed, submerged in a 42oC water bath and incubated for 12 to 18 h. Subsequently, each array was washed in 1X SSC and 0.2% SDS for two minutes, 0.1X SSC and 0.2% SDS for two minutes, and 0.1X SSC for two minutes, followed by a quick rinse in autoclaved, Millipore water. Arrays were then dried via centrifugation. This protocol is also available at our project web site:
http://maize-meristems.plantgenomics.iastate.edu/resources/protocols/
Scan protocol Fluorescence of Cy3 and Cy5 on slides was detected at 10-micron resolution with a ScanArray 5000 (Packard). The following setting was used as initial laser power and PMT gain, respectively: 82 and 63 for Cy3 and 77 and 58 for Cy5. These values were slightly adjusted depending on slides to have enough signals for the lowest scans and to have approximately the same amounts of signals between the two channels for the majority of the spots. For each successive scan, laser power and PMT gain were increased by two to three units and three to four units, respectively. Each slide was scanned seven times. Subsequently, three scans for each slide (“low/L”, “medium/M” and “high/H”) were selected for statistical analyses. In details “L” means Low laser power and PMT, “M” means Medium laser power and PMT and “H” means High laser power and PMT.
Description RNA was isolated from LCM-collected L1 and L2 cells that were derived from five to ten SAMs to generate one biological replication. PicoPure RNA Isolation Kit (Arcturus/Molecular Devices) was used for RNA isolation as per manufacturer’s instruction. In total, six biological replications were used for each microarray platform (see below). Approximately 1 ng of total RNA from each of the L1 and L2 RNA samples was used as starting material for RNA amplification. RiboAmp HS RNA Amplification Kit (Arcturus/Molecular Devices) was used as per manufacturer’s instruction. Each RNA sample yielded 30 to 90 g of amplified RNA (aRNA). Five to ten micrograms of each aRNA sample (the same amount of aRNA was used within each replication) were indirectly labeled with Cy dye and hybridized to the GPL2557, GPL2572, and GPL3538 sample platforms.
Data processing Spots were removed for bad PCR and empty on the microarrays. All data were background corrected and then an R implementation of the lowess normalization method (Dudoit and Fridlyand, 2002, Genome Biol: 3 RESEARCH0036) was used to normalize the two channels for each combination of slide and scan intensity. The data were centered around 0 for each slide. Subsequently, the data from each scan was used to conduct a mixed linear model analysis separately for each of 44,654 spots using a strategy similar to that of Wolfinger et al. (2001, J Comput Biol 8: 625-637). Following the statistical analysis, an additional 6,608 spots were removed from the data set because of concerns regarding the quality of the associated DNA sequences; 384 control spots that contained exogenous DNA were also removed. As a result, this study reports the gene expression patterns of 37,662 “informative” spots.
 
Submission date Nov 13, 2007
Last update date Nov 15, 2007
Contact name Patrick S. Schnable
E-mail(s) schnable@iastate.edu
Phone 515-294-0975
Organization name Iowa State University
Street address 2035B Roy J Carver Co-Lab
City Ames
State/province IA
ZIP/Postal code 50011
Country USA
 
Platform ID GPL2557
Series (1)
GSE9610 A global analysis of gene expression in histological layers of the shoot apical meristem of maize

Data table header descriptions
ID_REF
VALUE Normalized log ratio value of background corrected and median centered intensities of red channel and green channel
Ch1_Signal Mean Pixel intensity averaged over the local signal region for green channel (Cy3)
Ch1_Background Mean Pixel intensity averaged over the local background region for green channel (Cy3)
Ch1_Signal Median Median pixel intensity computed over the local signal region for green channel (Cy3)
Ch1_Background Median Median pixel intensity computed over the local background region for green channel (Cy3)
Ch2_Signal Mean Pixel intensity averaged over the local signal region for red channel (Cy5)
Ch2_Background Mean Pixel intensity averaged over the local background region for red channel (Cy5)
Ch2_Signal Median Median pixel intensity computed over the local signal region for red channel (Cy5)
Ch2_Background Median Median pixel intensity computed over the local background region for red channel (Cy5)
Ch1_Norm Background corrected, normalized and median centered log value of green channel(Cy3)
Ch2_Norm Background corrected, normalized and median centered log value of red channel(Cy5)

Data table
ID_REF VALUE Ch1_Signal Mean Ch1_Background Mean Ch1_Signal Median Ch1_Background Median Ch2_Signal Mean Ch2_Background Mean Ch2_Signal Median Ch2_Background Median Ch1_Norm Ch2_Norm
1 -0.335077243 2455.9592 79.7626 2386 0 1350.9166 172.3411 1282.5 61 0.317574525 -0.017502718
2 -0.216625109 3197.5214 110.7404 3127 0 1964.8485 192.8971 1796.5 81.5 0.579345349 0.36272024
3 -0.371424972 3471.375 106.8905 3250 0 1832.3038 208.1867 1787.5 81 0.66149498 0.290070008
4 -0.024890207 419.7187 125.7569 305 2 402.9687 184.2025 316 63 -1.387917761 -1.412807968
5 -0.306463672 1755.3164 97.2926 1671 0 1033.927 189.5217 927 71 -0.010631285 -0.317094957
6 -0.064320132 4493.375 142.5586 4583.5 9.5 3218.8076 225.4272 3116 112.5 0.922291097 0.857970965
7 -0.27896956 766 102.7601 705 7.5 530.0568 194.3468 422.5 92 -0.836728538 -1.115698098
8 -0.251596416 1087.8129 120.9085 1041 6 722.0183 233.9519 665 90 -0.490151264 -0.74174768
9 -0.045995104 4960.3115 99.5863 4882 0 3603.2604 183.9061 2953 92 1.024859927 0.978864823
10 0.042498019 1954.2857 101.963 1889.5 0 1576.5389 182.4375 1386 77 0.089828711 0.13232673
11 -0.163629772 4081.8725 97.1977 4173 0 2590.6623 160.5959 2568 43 0.825897 0.662267228
12 0.113840603 3169.642 96.997 3231.5 0 2689.8859 189.5603 2186 87.5 0.571350583 0.685191186
13 0.383300595 5615.4858 94.867 5846 0 6231.1928 205.6058 6344.5 78.5 1.152626369 1.535926964
14 -0.09633868 1092.897 89.6615 1014.5 0 799.5833 147.2695 652.5 53 -0.479514751 -0.575853431
15 -0.336769721 1585.0939 111.0149 1512 5.5 918.9347 175.4186 820 73.5 -0.113178514 -0.449948235
16 -0.399315933 4372.0185 97.376 4410.5 0 2254.5959 191.6058 2206 102.5 0.893735503 0.49441957
17 -0.053147347 2463.3166 106.6848 2445.5 0 1800.0059 193.1795 1755 89.5 0.319115156 0.265967809
18 -0.482741171 2337.6 83.3822 2372 0 1156.931 201 1074 93 0.270499225 -0.212241946
19 -0.123899927 2944.6328 119.0812 2978 0 2011.0068 221.6477 1890.5 108 0.496989456 0.373089529
20 -0.120039024 4229.0297 109.4764 4216 1 2873.6313 210.9247 2820.5 111.5 0.862085491 0.742046467

Total number of rows: 19200

Table truncated, full table size 1623 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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