NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM88720 Query DataSets for GSM88720
Status Public on Jun 07, 2007
Title SAM vs seedling_3H
Sample type RNA
 
Channel 1
Source name Shoot apical meristem (SAM) and very young leaf primordia (P0 and P1). A SAM is comprised of pluripotent stem cells, which divide to regenerate themselves as well as to provide cells to form other organs such as leaves and stems.
Organism Zea mays
Characteristics Inbred line: B73
Developmental stage: Vegetative SAMs from 14-day old seedlings
Sample name: SAM3
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 830 to 860 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. After 14 days SAMs and seedlings were collected from the same batch of plants.
Extracted molecule total RNA
Extraction protocol The PicoPure RNA Isolation Kit (Arcturus) was used to extract total RNA from the maize SAMs collected with LCM 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. About 10 ng of the Dnase I-treated RNA samples were then amplified with T7 RNA polymerase-based (T7-based) RNA amplification according to the method of Nakazono et al. (2003, Plant Cell 15: 583-596) except that amplified RNA was purified with the RNeasy Mini Kit and that after the second strand synthesis cDNA was purified with the QIAquick PCR Purification Kit (Qiagen). The procedure is also available at our project website (http://maize-meristems.plantgenomics.iastate.edu/index.html).
Label Cy3
Label protocol Two micrograms of amplified RNA 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/index.html). To remove dye-specific effects in the statistical analyses, Cy dyes were swapped between the RNA samples with odd and even numbers.
 
Channel 2
Source name Above ground part of seedlings
Organism Zea mays
Characteristics Inbred line: B73
Developmental stage: 14-day old seedlings
Sample name: Seedling3
Growth protocol the same as ch1
Extracted molecule total RNA
Extraction protocol Six whole seedlings without roots (14-day old, 30 to 40 g) were ground in liquid nitrogen and mixed thoroughly with 360 mL of a solution consisting of 38% [v/v] phenol in saturated buffer (pH 4.3 + 0.2, Fisher Scientific), 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate [pH 5], 5% [v/v] glycerol. Half of the mixture (about 200 mL) was transferred to a 250 mL centrifuging tube and centrifuged at 12,000g at 4oC for 20 min. The cleared homogenate solution was transferred to a new tube and incubated at room temperature for 25 min. Then, 36 mL of chloroform was added and shaken vigorously for 15 sec followed by 10 min incubation at room temperature. The mixture was centrifuged at 12,000g at 4oC for 30 min and the aqueous phase (about 110 mL) was transferred to a new tube. Then, 36 ml of isopropanol and 36 mL of 0.8 M sodium citrate/1.2 M sodium chloride were added, mixed well and incubated at room temperature for 15 min followed by a centrifugation at 12,000g at 4oC for 20 min to have RNA pellet at the bottom of the tube. The RNA pellet was washed with 75% ethanol and the tube was centrifuged again at 7,500g at 4oC for 8 min. After removing the ethanol, the pellet was air-dried for 30 min at room temperature. The RNA was resuspended with 1.8 mL of diethyl pyrocarbonate (DEPC) treated water at 60oC for 15 min. The RNA solution was transferred into two 1.7-mL Eppendorf tubes and centrifuged at 12,000g at 4oC for 5 min. The supernatant was transferred to new tubes. Five to 30 g of the RNA samples were taken and cleaned up according to the RNA purification step in the RNA amplification procedure (see below). Ten nano-gram of the cleaned up RNA samples were then amplified with T7 RNA polymerase-based (T7-based) RNA amplification according to the method of Nakazono et al. (2003, Plant Cell 15: 583-596) except that amplified RNA was purified with the RNeasy Mini Kit and that after the second strand synthesis cDNA was purified with the QIAquick PCR Purification Kit (Qiagen). The procedure is also available at our project website (http://maize-meristems.plantgenomics.iastate.edu/index.html). Seeding1.1 and Seedling1.2, and Seedling4.1 and Seedling4.2, and Seedling6.1 and Seedling6.2 were amplified from the same RNA pools, respectively.
Label Cy5
Label protocol the same as ch1
 
 
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 hybridization procedure is also available at our project website (http://maize-meristems.plantgenomics.iastate.edu/index.html).
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”, “medium” and “high” laser power and PMT) were selected for statistical analyses.
Description RNA was isolated from six to ten LCM-collected SAMs using PicoPure RNA Isolation Kit (Arcturus) as per manufacturer’s instructions to generate one biological replication. RNA was also isolated from six above-ground portions of seedlings using “home made Trizol” solution (see full protocol description of extract for details) to generate one biological replication. In total, six biological replications were prepared. Approximately 10 ng of total RNA from each biological replication were used as starting material for T7-based linear RNA amplification, performed as described by Nakazono et al. (2003, Plant Cell 15: 583-596). Each biological replication yielded between 10 and 50 g of amplified RNA (aRNA). Two micrograms of aRNA for each sample was indirectly labeled with Cy dye and hybridized to the GPL2557 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 18,302 spots using a strategy similar to that of Wolfinger et al. (2001, J Comput Biol 8: 625-637). By following the statistical analysis, an additional 3,710 spots were removed from the data set because of concerns regarding the quality of the associated DNA sequences and 192 exogenous spots also were removed. As a result, this study report the gene expression patterns of 14,400 informative spots
 
Submission date Dec 20, 2005
Last update date Jun 06, 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 (3)
GSE4004 Global gene expression in the maize shoot apical meristem
GSE4722 Global gene expression analysis in the shoot apical meristem of maize
GSE6267 A global gene expression analysis in the shoot apical meristem of maize (Zea mays L.)

Data table header descriptions
ID_REF
VALUE -[INV_VALUE]
Ch1_Signal Mean Pixel intensity avaeraged over the local signal region for green channel (Cy3)
Ch1_Background Mean Pixel intensity avaeraged 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 avaeraged over the local signal region for red channel (Cy5)
Ch2_Background Mean Pixel intensity avaeraged 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 and normalized log value of green channel(Cy3)
Ch2_norm Background corrected and normalized log value of red channel(Cy5)
INV_VALUE Normalized log ratio value of background corrected intensities of red channel and green channel

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 INV_VALUE
1 0.070785 8463.9492 1115.3273 7344.5 577 7289.1328 248.946 7612.5 136 8.905237 8.834452 -0.070785
2 1.19623 17116.5136 904.8623 16533 636 6249.3857 233.3989 5963 153.5 9.768802 8.572568 -1.196234
3 0.022829 11360.7333 1005.026 11321 572 12891.0117 308.1242 13173 225.5 9.387113 9.364284 -0.022829
4 -0.504707 1175.9721 935.0897 1018.5 663 1001.7404 319.6936 948 269 5.94686 6.451567 0.504707
5 0.003348 7737.687 918.2815 6806 680.5 7016.1977 273.0826 7371 190 8.80153 8.798182 -0.003348
6 0.714075 15078.6113 781.9553 14758.5 535.5 8313.5585 245.7155 8707.5 188.5 9.663466 8.949391 -0.714075
7 -0.714145 2180.6054 838.309 2018 581 2981.4873 229.5358 2865 168 7.228565 7.94271 0.714145
8 1.2031 4784.4375 923.4086 4348 682.5 1443.0909 243.0297 1207 195.5 8.165134 6.962037 -1.203097
9 0.732256 6252.1494 856.1156 5358.5 602 3446.3701 242.9034 2509 185.5 8.475497 7.743241 -0.732256
10 0.549905 9354.247 818.4578 5648.5 562.5 4964.2592 246.4506 3288 173 8.564327 8.014422 -0.549905
11 0.368547 14022.5693 951.2207 12708 605.5 9796.664 243.1239 10432 178 9.502659 9.134112 -0.368547
12 -0.210162 7871.3378 903.0369 7510 578.5 9455.6611 301.3742 10475 212 8.935106 9.145268 0.210162
13 0.584413 10993.6308 1091.9083 9820 696 5912.4501 300.6539 6226.5 174 9.205789 8.621376 -0.584413
14 0.802978 6454.0151 856.3466 4500 657 3699.281 262.4818 1776 147 8.226791 7.423813 -0.802978
15 0.769109 6574.7998 1124.3205 5590 580 2810.5187 369.9948 2559 179 8.531888 7.762779 -0.769109
16 0.438098 16029.538 1053.4061 14145 701 11722.2099 336.9281 10902.5 193 9.61172 9.173622 -0.438098
17 0.603549 7120.25 860.7341 6942 633 4507.1303 364.6444 4068 202 8.806837 8.203288 -0.603549
18 2.11962 10207.416 1076.2861 9762 758 1534.7208 281.4205 1269 187 9.106323 6.986703 -2.11962
19 0.103027 3429.3371 1190.9421 2180 782.5 2351.5449 504.3272 1449 176 7.24805 7.145023 -0.103027
20 0.010182 2255.5261 1691.9602 1880 731 1523.2946 250.2099 1380 181 7.073888 7.063706 -0.010182

Total number of rows: 19200

Table truncated, full table size 1847 Kbytes.




Supplementary data files not provided

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap