NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM88633 Query DataSets for GSM88633
Status Public on Jun 07, 2007
Title SAM vs seedling_1H
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: SAM1
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: Seedling1.1
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 0.087006 2614.1738 313.4356 2519 196 1705.8312 42.2989 1769 0 7.658393 7.571387 -0.087006
2 1.28356 9540.2275 333.9224 9931 164 2294.2292 44.047 2419 0 9.130975 7.847415 -1.28356
3 0.186847 9599.3515 250.5706 9661 116 6811.9169 47.7535 7379 0 9.128627 8.94178 -0.186847
4 -2.69703 259.055 216.7823 123 118 112.4678 52.6438 56 0 1.56889 4.265921 2.697031
5 -0.180221 3108.1337 292.3137 2964.5 122 2724.7517 53.8053 2899 0 7.872518 8.052739 0.180221
6 0.470483 5548.0087 321.1171 5552 175 2813.6884 46.383 2931.5 0 8.522083 8.0516 -0.470483
7 -0.915349 859.1101 270.5516 659 144 1294.8073 50.8261 1150 0 6.189572 7.104921 0.915349
8 0.939554 2132.8544 229.0353 1412.5 110 558.612 41.9384 454 0 7.11633 6.176776 -0.939554
9 0.850898 5187.5908 271.4937 5017.5 133 1792.9514 39.1744 1780.5 0 8.415068 7.56417 -0.850898
10 0.404076 3201.9663 260.3792 2926 127 1632.9603 51.8559 1553.5 0 7.84518 7.441104 -0.404076
11 0.565432 10273.0136 447.8379 9616 249.5 5033.7153 231.7034 4998 48.5 9.108839 8.543407 -0.565432
12 -0.315152 9362.2861 619.3383 8678 367 10567.9062 390.4923 10859 233.5 8.990705 9.305857 0.315152
13 0.09097 5985.1562 353.32 5986 196 4546.0893 118.6532 4792 12.5 8.613665 8.522695 -0.09097
14 0.361273 2805.4094 245.4201 2793 69 1603.6845 67.2175 1578 0.5 7.817863 7.45659 -0.361273
15 0.691508 2060.7033 285.085 1903.5 165.5 758.9606 41.9023 727 0 7.371437 6.679929 -0.691508
16 0.369069 10596.6972 281.1405 10772.5 104 6361.6093 53.3833 6875 0 9.240003 8.870934 -0.369069
17 0.658279 4787.122 263.623 4642 150 2100.3488 66.1815 1988 0 8.331971 7.673692 -0.658279
18 2.16792 6031.6577 259.5733 6155 104.5 607.1205 49.0387 573 0 8.614303 6.446387 -2.167916
19 0.161923 1149.6137 272.5955 935 146 701.7 59.1292 607.5 0 6.622476 6.460553 -0.161923
20 0.498769 859.0155 220.6344 690 99 393.1666 46.0765 330 0 6.342197 5.843428 -0.498769

Total number of rows: 19200

Table truncated, full table size 1783 Kbytes.




Supplementary data files not provided

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