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Sample GSM88716 Query DataSets for GSM88716
Status Public on Jun 07, 2007
Title SAM vs seedling_2M
Sample type RNA
 
Channel 1
Source name Above ground part of seedlings
Organism Zea mays
Characteristics Inbred line: B73
Developmental stage: 14-day old seedlings
Sample name: Seedling2
Treatment 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.
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 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 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 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: SAM2
Growth protocol the same as ch1
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 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 Normalized log ratio value of background corrected intensities of red channel and green channel
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)

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 0.045174 2119.0559 196.3117 2092 138.5 2444.8662 240.2023 2364 185 7.609898 7.655072
2 0.449799 3310.6284 189.6483 3301 136 4974.2534 294.1383 5169.5 211 8.059743 8.509542
3 -0.056117 6296.6303 183.5615 6857 142 5704.5712 226.3623 6242 178 8.789327 8.73321
4 -0.12807 206.0366 198.0501 175 150 240.2385 227.1169 217 189 3.376731 3.248661
5 -0.323645 2947.6889 244.3362 2968 174.5 2342.4924 262.6837 2339.5 228 7.95734 7.633695
6 0.513018 3228.4426 185.0497 3118 120.5 4956.374 195.1756 5183 159 8.007515 8.520533
7 -1.129934 1271.9796 206.7083 1195 156.5 579.1547 211.3636 566 178.5 7.019361 5.889427
8 1.039988 509.6054 263.4773 475 229 1094.6999 269.9624 1070 255.5 5.586601 6.626589
9 0.487764 2425.0317 321.0865 2386.5 269 3772.9243 341.1193 3851 280 7.67579 8.163554
10 -0.577167 1656.4359 278.4401 1454 209 1030.9392 251.0038 1003 203 7.195361 6.618194
11 -2.790865 4775.7719 324.0953 4924 257.5 564 243.5105 532 205 8.516129 5.725264
12 -1.503284 11617.1503 336.3588 13125.5 202 2862.3549 279.7982 2938 210 9.440927 7.937643
13 -0.548672 4003.455 216.092 4014 163 2448.8261 229.5931 2485 194 8.2711 7.722428
14 -0.641623 1985.1195 201.1556 1949 153 1324.3809 233.7278 1267.5 196 7.556622 6.914999
15 0.141586 1154.03 202.7277 1120 147 1482.2846 209.0435 1456 170 6.949947 7.091533
16 -0.90107 9520.7099 253.5245 9596 181 4183.2377 245.3531 3843 211 9.124525 8.223455
17 0.188857 1823.4774 251.0121 1756 221 2296.7624 276.0925 2265 239 7.381196 7.570053
18 1.247214 823.3525 261.3841 784 221 2448.101 259.5887 2465.5 215 6.403596 7.65081
19 -0.251976 756.9393 200.0702 655 156 686.2616 217.1961 638.5 187 6.290686 6.03871
20 0.142012 456.7267 381.9523 408.5 243 631.4705 411.1064 502.5 276 5.200067 5.342079

Total number of rows: 19200

Table truncated, full table size 1629 Kbytes.




Supplementary data files not provided

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