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Sample GSM88724 Query DataSets for GSM88724
Status Public on Jun 07, 2007
Title SAM vs seedling_5L
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: SAM5
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: Seedling5
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 0.163223 470.705 39.2641 398 0 242.1933 3.0448 230.5 0 5.798382 5.635159 -0.163223
2 1.63292 1648.1718 50.2783 1640.5 0 255.125 3.6891 248 0 7.27687 5.643949 -1.632921
3 0.288318 1241.5692 39.9623 1239 0 740.7832 2.948 758 0 7.021593 6.733275 -0.288318
4 -0.122328 36.3387 24.3502 0 0 3.5887 2.8315 0 0 -0.06116412 0.06116412 0.12232824
5 0.021302 353.8467 28.2842 325.5 0 237.8394 3.4693 213 0 5.587854 5.566552 -0.021302
6 0.416209 679.2601 32.2892 675 0 345.471 2.7936 328 0 6.36423 5.948021 -0.416209
7 -1.17594 102.0366 36.0453 45 0 90.9449 2.3747 60 0 3.381786 4.557729 1.175943
8 2.43288 147.3302 38.105 106 0 16.7155 2.9785 0 0 3.552852 1.119977 -2.432875
9 1.41273 484.725 34.272 448 0 77.4128 5.607 69 0 5.884122 4.471396 -1.412726
10 0.988705 270.1475 43.6928 230.5 0 72.2741 9.6148 47.5 0 5.157424 4.168719 -0.988705
11 0.773475 1070.8057 35.9128 1116 0 414.5338 5.4638 403 0 6.896646 6.123171 -0.773475
12 0.143138 894.3816 33.2453 863 0 577.4637 2.9429 593.5 0 6.646216 6.503078 -0.143138
13 0.641479 618.937 35.9299 616 0 233.197 3.2023 230 0 6.254383 5.612904 -0.641479
14 2.09789 312.3511 31.534 269 0 45.1626 7.6074 14 0 5.202181 3.104291 -2.09789
15 3.41633 291.6062 24.7301 241 0 23.3027 2.5518 0 0 4.452636 1.036302 -3.416334
16 0.766094 912.0694 25.0416 907 0 320.3006 2.1955 317 0 6.669695 5.903601 -0.766094
17 1.23965 592.3023 31.8585 544 0 128.1612 3.8493 103.5 0 6.09481 4.855163 -1.239647
18 4.342 557.8231 30.3454 508 0 9.5862 1.9861 0 0 5.287226 0.9452218 -4.3420042
19 2.44239 139.1379 35.252 77 0 17.0214 4.5109 0 0 3.399551 0.9571581 -2.4423929
20 2.34519 127.8197 29.014 91 0 12.7176 2.4301 0 0 3.433488 1.088301 -2.345187

Total number of rows: 19200

Table truncated, full table size 1629 Kbytes.




Supplementary data files not provided

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