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Sample GSM4559 Query DataSets for GSM4559
Status Public on Jul 25, 2003
Title wholeRefCy3 vs CNS1Cy5 610620GEO
Sample type RNA
 
Channel 1
Source name Central nervous system at approximately 18 hours before pupariation
Organism Drosophila melanogaster
Extracted molecule total RNA
 
Channel 2
Source name A reference sample from whole animal at approximately 18 hours before pupariation
Organism Drosophila melanogaster
Extracted molecule total RNA
 
 
Description The MIAME Checklist
Experiment Design:
· Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
Comparisons between individual tissues/organs at approximately 18 hours before pupariation staged according to Andres, A.J. & Thummel, C.S. Methods for quantitative analysis of transcription in larvae and prepupae. in Methods in Cell Biology, Volume 44. Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994)) vs. a reference sample from identically staged whole animal. Abbreviations are: MG, midgut; SG, salivary gland; ED, epidermis with attached muscle; CNS, central nervous system; WD, wing disc. RNA samples from Drosophila melanogaster (Canton S strain) were amplified according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29. Samples from one tissue/organ were hybridized against the reference sample on each microarray.
· Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
Each array tested differences in gene expression in different tissues/organs at one developmental stage.
· The number of hybridizations performed in the experiment.
Three replicate hybridizations for each tissue using independent samples.
· The type of reference used for the hybridizations, if any.
The reference sample is from identically staged whole animal.
· Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
See above
· Quality control steps taken: for example, replicates or dye swaps.
No dye swaps were used. Three replicates for each tissue/organ.
· URL of any supplemental websites or database accession numbers
Samples used, extract preparation and labeling:
· The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state. See above. Tissues/Organs were dissected from at least 12 individuals for each sample.
· Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques. Animals were grown on standard corn meal medium with 0.05% bromophenol blue at 25 degrees Celsius.
· Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol
Total RNA was extracted using the SDS lysis protocol in Andres, A.J. & Thummel, C.S. “Methods for quantitative analysis of transcription in larvae and prepupae”. in Methods in Cell Biology, Volume 44. Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994). 2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.
· Labeling protocol(s).
Priming step with random hexamers. Labeling step using Superscript II, buffer, DTT, dUTP-Cy3, dUTP-Cy5 and free nucleotides. Cleanup using Qiagen PCR cleanup kit. Hybridization to slide in a TE, SSC, SDS solution with poly-A as a blocker. Hybridization chambers from DIE-TECH, incubated for 10 minutes at 42 degrees followed by approximately 15-hour incubation at 64 degrees. Three washes with successively more dilute solutions of SSC, including SDS in the first wash, then a centrifuge spin to dry. Arrays were stored in the dark until scanned. See K.P. White and K.C. Burtis “Drosophila microarrays: from arrayer construction to hybridization” in Drosophila Protocols, Cold Spring Harbor Press, 2000 for microarray protocols.
· External controls (spikes).
Hybridization procedures and parameters:
· The protocol and conditions used during hybridization, blocking and washing.
See above.
Measurement data and specifications:
· The quantitations based on the images.
Images were gridded and spots were quantified with GenePix software (Axon Instruments)
· The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
Slides were scanned on an Axon 4000B scanner using Genepix Pro 3.0 software.
o Type of image analysis software used: specifications should be stated in the materials and methods.
The program GenePix Pro 3.0 from Axon Instruments was used to analyse the data.
o A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
(F635 Median - B635), (F532 Median - B532), and (Ratio of Medians) were the raw measurements produced by the software. The detailed information for these measurements is available at http://www.axon.com/manuals/GenePix_Pro_4.0_User_Guide_Rev_E.pdf
o The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
See *.xls files attached.
o Data selection and transformation procedures.
Data for all spots are filtered. For a spot to pass quality control, 3 conditions need to be met: (1). There is no automatic or manual flag during image gridding using GenePix Pro software. (2). For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background. (3). The net signal noise ratio should be at least 1 for at least one channel. For the channel 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) to the background median. The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot. For the channel 532 nm wavelength, F532_median and B532_median are used. Ratios between net signals are log2 transformed and analyzed as detailed in the paper.
o Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
See the web tables attached for the paper. Additionally, see the column for Passed and normalized ratio of median in the *.xls files attached. Data before and after data selection is in one file.
Array Design:
· General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known – often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
Spotted array using PCR fragments of approximately 95% of the genes in the D. melanogaster BDGP genome. Slides were coated with polylysine, printed using a GeneMachines OmniGrid printer with DNA suspended in 3X SSC, and postprocessed using the DCE protocol of Diehl, F., Grahlmann, S., Beier, M. & Hoheisel, J.D. “Manufacturing DNA microarrays of high spot homogeneity and reduced background signal.” Nucleic Acids Research 29, e38 (2001).
· For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The KP numbers identify these and can be tied to the data files. The ID of the respective reporter is included in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
· For each reporter,
 
Submission date Feb 24, 2003
Last update date Nov 21, 2005
Contact name Tong-Ruei Li
E-mail(s) [email protected]
Phone 203-785-4474
Organization name Yale University School of Medicine
Department Department of Genetics
Lab Kevin White
Street address 333 Cedar St. Rm#NSB386
City New Haven
State/province CT
ZIP/Postal code 06511
Country USA
 
Platform ID GPL248
Series (1)
GSE541 Whole animal vs. tissues at 18h before puparium formation

Data table header descriptions
ID_REF
X X-coordinate of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image
Y Y-coordinate of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image
Dia. Diameter in �m of the feature-indicator
F635 Median Median pixel intensity of feature for Cy5 channel
F635 Mean Mean pixel intensity of feature for Cy5 channel
F635 SD Cy5 pixel intensity standard deviation
B635 Median Median Cy5 feature background intensity
B635 Mean Mean Cy5 feature background intensity
B635 SD Cy5 background pixel intensity standard deviation
% > B635+1SD Percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #1 (635 nm, Cy5)
% > B635+2SD Percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #1 (635 nm, Cy5)
F635 % Sat. Percentage of pixel saturation within feature
F532 Median Median feature pixel intensity at wavelength #2 (532 nm, Cy3)
F532 Mean Mean feature pixel intensity at wavelength #2 (532 nm, Cy3)
F532 SD Standard deviation of the feature pixel intensity at wavelength #2 (532 nm, Cy3)
B532 Median Median feature background intensity at wavelength #2 (532 nm, Cy3)
B532 Mean Mean feature background intensity at wavelength #2 (532 nm, Cy3)
B532 SD Standard deviation of the feature background intensity at wavelength #2 (532 nm, Cy3)
% > B532+1SD Percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #2 (532 nm, Cy3)
% > B532+2SD Percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #2 (532 nm, Cy3)
F532 % Sat. Percentage of feature pixels at wavelength #2 (Cy3) that are saturated
Ratio of Medians Ratio of the background subtracted median pixel intensity at the second wavelength (Cy3), to the background subtracted median pixel intensity at the first wavelength(Cy5)
Ratio of Means Ratio of the arithmetic mean of the background subtracted raw pixel intensities at the second wavelength (Cy3), to the arithmetic mean of the background subtracted raw pixel intensities at the first wavelength (Cy5)
Median of Ratios Median of the pixel-by-pixel ratios of pixel intensities that have had the median background intensity subtracted of wavelength 2 (Cy3) to wavelength one (Cy5)
Mean of Ratios Arithmetic mean of the pixel-by-pixel ratios of the raw pixel intensities of wavelength 2 (Cy3) to wavelength 1 (Cy5)
Ratios SD Standard deviation of the log of pixel intensity ratios
Rgn Ratio Regression ratio is determined by computing a linear regression between the population of pixels represented by wavelength 1 and wavelength 2
Rgn R� Coefficient of determination provides a measure of the level of accuracy of the fit of the linear regression curve
F Pixels Number of feature pixels
B Pixels Number of background pixels
Sum of Medians Sum of the median of the pixel intensities at each wavelength, with the median background pixel intensity at each wavelength subtracted
Sum of Means Sum of the arithmetic mean of the pixel intensities at each wavelength, with the median background pixel intensity at each wavelength subtracted
Log Ratio Log Ratio
F635 Median - B635 Cy5 median feature intensity subtracted by Cy5 median background intensity
F532 Median - B532 Cy3 median feature intensity subtracted by Cy3 median background intensity
F635 Mean - B635 Cy5 mean feature intensity subtracted by Cy5 mean background intensity
F532 Mean - B532 Cy3 mean feature intensity subtracted by Cy3 mean background intensity
Flags A value of -100 means that this spot was bad. A value of -75 means that the ID for this spot is empty. A value of -50 means that this spot cannot be aligned during analysis. A value of 0 or larger than 0 means that this spot passed the flagging screen but may still fail other quality tests.
NormFactorMedian The normalization factor calculated using Ratio of Medians
VALUE same as UNF_VALUE but with flagged values removed
QUALITY For a spot to pass quality control, 3 conditions need to be met: 1. There is no automatic or manual flag during image gridding using GenePix Pro software. 2. For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background. 3. The net signal noise ratio should be at least 1 for at least one channel. For the channel 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) and the background median. The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot. For the channel 532 nm wavelength, F532_median and B532_median are used.
PRE_VALUE The product of Ratio of Medians and NormFactorMedian
UNF_VALUE log2 ratio (log2 of PRE_VALUE)

Data table
ID_REF X Y Dia. F635 Median F635 Mean F635 SD B635 Median B635 Mean B635 SD % > B635+1SD % > B635+2SD F635 % Sat. F532 Median F532 Mean F532 SD B532 Median B532 Mean B532 SD % > B532+1SD % > B532+2SD F532 % Sat. Ratio of Medians Ratio of Means Median of Ratios Mean of Ratios Ratios SD Rgn Ratio Rgn R� F Pixels B Pixels Sum of Medians Sum of Means Log Ratio F635 Median - B635 F532 Median - B532 F635 Mean - B635 F532 Mean - B532 Flags NormFactorMedian VALUE QUALITY PRE_VALUE UNF_VALUE
1 2800 16250 100 211 219 51 213 215 44 22 6 0 179 194 61 164 167 32 36 21 0 -0.133 0.2 0.8 0.866 3.079 1.311 0.039 80 473 13 36 Error -2 15 6 30 -75 1.04522 0 -0.13901426
2 2970 16250 100 234 260 121 216 218 43 37 21 0 184 216 107 162 167 32 45 30 0 0.818 0.815 1.383 1.447 4.191 1.517 0.132 80 443 40 98 -0.29 18 22 44 54 -75 1.04522 0 0.85498996 -0.2260213
3 3140 16250 100 226 246 117 220 220 44 22 13 0 186 208 116 163 165 32 43 21 0 0.261 0.578 1.222 1.135 4.279 1.084 0.197 80 442 29 71 -1.939 6 23 26 45 -75 1.04522 0 0.27280242 -1.8740723
4 3310 16250 100 231 247 79 225 228 43 23 15 0 188 212 80 171 172 32 42 21 0 0.353 0.537 1.5 1.315 4.959 1.331 0.039 80 442 23 63 -1.503 6 17 22 41 -75 1.04522 0 0.36896266 -1.4384533
5 3480 16250 100 229 237 64 231 234 42 23 8 0 182 194 61 175 178 31 28 20 0 -0.286 0.316 1.346 1.288 3.536 2.305 0.06 80 442 5 25 Error -2 7 6 19 -75 1.04522 0 -0.29893292
6 3650 16250 100 229 242 61 237 237 44 21 8 0 193 210 71 179 181 31 37 20 0 -0.571 0.161 1.194 1.227 3.608 1.087 0.077 80 442 6 36 Error -8 14 5 31 -75 1.04522 0 -0.59682062
7 3820 16250 100 243 256 68 249 252 45 22 12 0 203 226 105 187 189 34 33 17 0 -0.375 0.179 0.984 1.008 3.317 0.804 0.182 80 454 10 46 Error -6 16 7 39 -75 1.04522 0 -0.3919575
8 4000 16250 100 261 269 67 259 263 48 18 13 0 202 225 87 193 197 52 26 12 0 0.222 0.313 1.103 1.032 3.644 0.664 0.055 80 454 11 42 -2.17 2 9 10 32 -75 1.04522 0 0.23203884 -2.1075623
9 4170 16250 100 270 283 79 269 271 50 22 13 0 202 229 86 196 198 36 31 15 0 0.167 0.424 0.865 0.799 3.681 1.336 0.139 80 442 7 47 -2.585 1 6 14 33 -75 1.04522 0 0.17455174 -2.5182733
10 4340 16250 100 276 280 69 273 277 51 18 7 0 205 229 94 199 200 35 30 16 0 0.5 0.233 0.978 1.112 4.69 0.611 0.021 80 442 9 37 -1 3 6 7 30 -75 1.04522 0 0.52261 -0.9361933
11 4510 16250 100 280 292 81 282 285 50 25 12 0 221 241 84 202 203 34 36 22 0 -0.105 0.256 1.229 1.165 3.901 1.309 0.043 80 432 17 49 Error -2 19 10 39 -75 1.04522 0 -0.1097481
12 4680 16250 100 286 302 93 292 293 60 21 7 0 225 249 103 203 205 34 36 20 0 -0.273 0.217 1.172 0.986 3.373 1.663 0.124 80 442 16 56 Error -6 22 10 46 -75 1.04522 0 -0.28534506
13 4850 16250 100 293 321 103 297 298 51 28 16 0 221 273 144 210 214 36 32 23 0 -0.364 0.381 1.75 1.336 4.165 0.625 0.236 80 442 7 87 Error -4 11 24 63 -75 1.04522 0 -0.38046008
14 5020 16250 100 353 450 341 307 308 58 37 26 0 244 335 336 221 230 80 20 15 0 2 1.254 2.079 1.613 3.685 0.851 0.245 80 442 69 257 1 46 23 143 114 -75 1.04522 0 2.09044 1.0638073
15 5190 16250 100 303 338 116 308 306 51 23 16 0 241 295 155 226 228 36 32 23 0 -0.333 0.435 1.083 1.261 3.724 0.659 0.142 80 442 10 99 Error -5 15 30 69 -75 1.04522 0 -0.34805826
16 5360 16250 100 312 360 147 311 310 51 30 18 0 238 298 177 229 232 37 36 22 0 0.111 0.71 1.026 1.019 3.477 0.881 0.328 80 432 10 118 -3.17 1 9 49 69 -75 1.04522 0 0.11601942 -3.1075623
17 5530 16250 100 325 354 111 317 321 54 28 20 0 251 296 131 236 238 37 36 28 0 0.533 0.617 1.125 1.173 3.373 0.993 0.22 80 442 23 97 -0.907 8 15 37 60 -75 1.04522 0 0.55710226 -0.8439863
18 5700 16250 100 312 369 176 323 327 54 28 18 0 248 307 181 240 244 41 25 18 0 -1.375 0.687 1.233 1.676 3.394 1.003 0.312 80 432 -3 113 Error -11 8 46 67 -75 1.04522 0 -1.4371775
19 5870 16240 100 341 374 134 338 341 58 25 12 0 267 321 173 242 246 41 37 23 0 0.12 0.456 0.78 0.998 3.337 0.689 0.273 80 437 28 115 -3.059 3 25 36 79 -75 1.04522 0 0.1254264 -2.9950873
20 6040 16240 100 336 376 134 336 349 109 18 11 0 268 319 152 243 259 104 25 15 0 0 0.526 0.864 0.804 3.793 0.819 0.259 80 428 25 116 Error 0 25 40 76 -75 1.04522 0 0

Total number of rows: 20000

Table truncated, full table size 3900 Kbytes.




Supplementary data files not provided
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