NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM4491 Query DataSets for GSM4491
Status Public on Jul 25, 2003
Title wholeRefCy3 vs ED2Cy5 700600GEO
Sample type RNA
 
Channel 1
Source name Epidermis with attached muscle 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 2750 16850 100 332 677 691 308 319 95 40 32 0 160 306 295 131 134 26 50 41 0 0.828 2.109 2.356 2.362 4.216 2.914 0.544 80 475 53 544 -0.273 24 29 369 175 -75 1.11756 0 0.92533968 -0.1119453
2 2920 16850 100 341 568 525 316 328 94 41 28 0 147 228 173 134 140 36 35 27 0 1.923 2.681 3.649 3.609 3.613 3.782 0.557 80 462 38 346 0.943 25 13 252 94 -75 1.11756 0 2.14906788 1.1037113
3 3090 16850 100 329 410 238 321 329 95 31 22 0 146 197 124 134 137 30 40 26 0 0.667 1.413 1.944 1.775 3.884 3.029 0.335 80 453 20 152 -0.585 8 12 89 63 -75 1.11756 0 0.74541252 -0.4238893
4 3260 16850 100 349 379 199 343 347 96 22 16 0 158 199 132 136 138 30 42 28 0 0.273 0.571 1.731 1.696 4.621 3.183 0.152 80 440 28 99 -1.874 6 22 36 63 -75 1.11756 0 0.30509388 -1.7126753
5 3430 16850 100 353 375 159 345 356 99 27 12 0 154 187 97 136 139 34 37 27 0 0.444 0.588 1.793 1.683 4.5 4.378 0.135 80 461 26 81 -1.17 8 18 30 51 -75 1.11756 0 0.49619664 -1.0110163
6 3600 16850 100 343 364 146 344 343 94 25 12 0 140 167 68 135 137 30 37 25 0 -0.2 0.625 2.403 2.528 3.97 5.914 0.119 80 444 4 52 Error -1 5 20 32 -75 1.11756 0 -0.223512
7 3770 16850 100 315 335 116 362 359 100 11 3 0 147 153 53 138 141 29 27 17 0 -5.222 -1.8 2.417 2.278 3.678 13.824 0.034 80 454 -38 -12 Error -47 9 -27 15 -75 1.11756 0 -5.83589832
8 3940 16850 100 378 393 122 367 372 101 25 6 0 173 186 74 145 147 27 50 27 0 0.393 0.634 1.931 1.697 4.632 8.681 0.042 80 440 39 67 -1.348 11 28 26 41 -75 1.11756 0 0.43920108 -1.1870463
9 4110 16850 100 353 398 196 348 362 100 22 13 0 173 202 93 142 144 26 55 36 0 0.161 0.833 1.602 1.932 4.015 5.22 0.138 80 440 36 110 -2.632 5 31 50 60 -75 1.11756 0 0.17992716 -2.4745153
10 4280 16850 100 372 427 186 365 367 91 28 20 0 149 200 112 140 143 29 42 32 0 0.778 1.033 2.492 3.089 3.862 3.488 0.19 80 453 16 122 -0.363 7 9 62 60 -75 1.11756 0 0.86946168 -0.2018063
11 4450 16850 100 388 424 140 381 385 101 26 12 0 161 201 106 144 145 29 43 31 0 0.412 0.754 2.25 2.119 4.127 3.809 0.115 80 440 24 100 -1.28 7 17 43 57 -75 1.11756 0 0.46043472 -1.1189313
12 4620 16850 100 419 484 251 394 399 107 28 18 0 168 239 166 148 154 40 40 31 0 1.25 0.989 1.677 1.672 3.965 2.083 0.39 80 461 45 181 0.322 25 20 90 91 -75 1.11756 0 1.39695 0.4822803
13 4790 16850 100 419 471 214 388 398 110 30 17 0 189 241 133 153 160 44 45 31 0 0.861 0.943 1.379 1.561 3.597 2.551 0.262 80 461 67 171 -0.216 31 36 83 88 -75 1.11756 0 0.96221916 -0.0555633
14 4960 16850 100 421 495 242 389 395 105 37 26 0 168 261 184 151 153 27 42 33 0 1.882 0.964 1.82 2.116 4.109 1.95 0.326 80 440 49 216 0.913 32 17 106 110 -75 1.11756 0 2.10324792 1.0726193
15 5130 16850 100 405 641 519 393 400 102 35 30 0 165 370 423 155 158 32 36 30 0 1.2 1.153 2.737 3.149 4.313 1.285 0.615 80 450 22 463 0.263 12 10 248 215 -75 1.11756 0 1.341072 0.4233873
16 5300 16850 100 441 825 782 378 389 93 45 33 0 179 548 705 158 160 30 45 33 0 3 1.146 2.167 2.584 3.479 1.106 0.669 80 451 84 837 1.585 63 21 447 390 -75 1.11756 0 3.35268 1.7453153
17 5470 16850 100 396 546 374 362 378 93 38 32 0 170 375 370 158 160 29 43 35 0 2.833 0.848 1.953 2.248 4.148 1.038 0.596 80 453 46 401 1.503 34 12 184 217 -75 1.11756 0 3.16604748 1.6626833
18 5640 16850 100 381 565 402 370 379 102 35 26 0 186 368 362 157 163 56 43 33 0 0.379 0.924 1.51 1.583 4.549 1.165 0.516 80 468 40 406 -1.399 11 29 195 211 -75 1.11756 0 0.42355524 -1.2393783
19 5810 16850 100 421 627 496 380 387 99 41 27 0 190 449 465 157 159 30 50 45 0 1.242 0.846 1.378 1.555 3.653 1.064 0.645 80 451 74 539 0.313 41 33 247 292 -75 1.11756 0 1.38800952 0.4730173
20 5980 16850 100 434 697 546 382 394 102 42 33 0 188 547 686 163 168 40 42 36 0 2.08 0.82 1.132 1.193 3.037 0.768 0.573 80 440 77 699 1.057 52 25 315 384 -75 1.11756 0 2.3245248 1.2169363

Total number of rows: 20000

Table truncated, full table size 3962 Kbytes.




Supplementary data files not provided
| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap