NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM4490 Query DataSets for GSM4490
Status Public on Jul 25, 2003
Title wholeRefCy3 vs ED1Cy5 740600GEO
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 2670 16230 100 436 578 429 359 372 119 40 26 0 164 242 180 141 143 24 46 38 0 3.348 2.168 2.751 2.749 4.569 3.931 0.312 80 464 100 320 1.743 77 23 219 101 -75 1.18786 0 3.97695528 1.9916643
2 2840 16230 100 432 539 323 343 358 111 47 26 0 161 226 158 145 147 27 42 30 0 5.563 2.42 2.208 2.653 3.334 3.133 0.436 80 439 105 277 2.476 89 16 196 81 -75 1.18786 0 6.60806518 2.7242283
3 3010 16230 100 411 561 509 379 382 122 31 16 0 153 294 381 150 153 41 23 20 0 10.667 1.264 2.148 2.32 2.87 1.568 0.577 80 446 35 326 3.415 32 3 182 144 -75 1.18786 0 12.67090262 3.6634473
4 3180 16230 100 417 468 229 393 402 114 30 16 0 162 244 207 149 150 26 45 28 0 1.846 0.789 1.929 3.012 3.517 1.848 0.237 80 439 37 170 0.885 24 13 75 95 -75 1.18786 0 2.19278956 1.1327673
5 3350 16230 100 472 520 273 403 413 129 30 18 0 171 254 215 156 158 28 43 28 0 4.6 1.194 3.391 2.937 3.687 2.136 0.252 80 439 84 215 2.202 69 15 117 98 -75 1.18786 0 5.464156 2.4499993
6 3520 16230 100 431 629 590 393 411 129 30 20 0 178 361 451 163 165 31 37 25 0 2.533 1.192 2.411 1.96 5.563 1.516 0.542 80 439 53 434 1.341 38 15 236 198 -75 1.18786 0 3.00884938 1.5892123
7 3690 16230 100 445 538 337 414 431 128 26 15 0 193 380 472 169 173 33 45 27 0 1.292 0.588 2.416 2.077 4.348 0.737 0.393 80 437 55 335 0.369 31 24 124 211 -75 1.18786 0 1.53471512 0.6179713
8 3860 16230 100 466 582 353 428 428 127 33 21 0 192 341 363 170 172 37 40 31 0 1.727 0.901 2.096 2.375 3.828 1.164 0.406 80 428 60 325 0.788 38 22 154 171 -75 1.18786 0 2.05143422 1.0366333
9 4030 16230 100 541 587 290 414 418 122 50 23 0 200 300 246 165 168 30 52 37 0 3.629 1.281 2.564 2.59 4.037 1.907 0.278 80 428 162 308 1.859 127 35 173 135 -75 1.18786 0 4.31074394 2.1079373
10 4200 16230 100 419 500 288 398 412 119 30 17 0 191 300 267 171 173 30 42 28 0 1.05 0.791 1.95 2.084 4.317 1.392 0.398 80 428 41 231 0.07 21 20 102 129 -75 1.18786 0 1.247253 0.3187543
11 4370 16240 100 421 488 246 390 395 117 32 18 0 185 283 266 171 174 33 37 26 0 2.214 0.875 2.247 2.002 4.011 1.36 0.263 80 432 45 210 1.147 31 14 98 112 -75 1.18786 0 2.62992204 1.3950203
12 4540 16240 100 453 506 222 378 391 117 41 23 0 191 276 210 171 175 33 43 27 0 3.75 1.219 1.39 1.95 4.653 1.665 0.265 80 453 95 233 1.907 75 20 128 105 -75 1.18786 0 4.454475 2.1552553
13 4710 16240 100 384 399 125 383 397 120 22 2 0 188 227 109 175 178 31 40 23 0 0.077 0.308 2.261 1.863 4.482 17.661 0.007 80 457 14 68 -3.7 1 13 16 52 -75 1.18786 0 0.09146522 -3.4506333
14 4890 16240 100 409 459 168 400 412 120 28 18 0 201 239 119 180 183 31 42 30 0 0.429 1 2.879 2.268 3.955 4.955 0.093 80 454 30 118 -1.222 9 21 59 59 -75 1.18786 0 0.50959194 -0.9725863
15 5060 16240 100 392 418 145 410 420 119 18 10 0 199 247 153 192 196 36 33 21 0 -2.571 0.145 2.619 2.633 3.476 2.78 0.088 80 453 -11 63 Error -18 7 8 55 -75 1.18786 0 -3.05398806
16 5230 16240 100 395 434 221 396 406 115 16 10 0 203 283 266 194 199 36 27 21 0 -0.111 0.427 1.733 1.851 4.372 1.18 0.269 80 432 8 127 Error -1 9 38 89 -75 1.18786 0 -0.13185246
17 5400 16240 100 410 454 213 392 400 116 28 11 0 207 307 234 197 200 35 37 31 0 1.8 0.564 1.765 1.654 4.87 1.325 0.217 80 453 28 172 0.848 18 10 62 110 -75 1.18786 0 2.138148 1.0963623
18 5570 16240 100 419 441 155 393 404 118 21 7 0 207 255 153 198 202 39 27 17 0 2.889 0.842 2.097 2.035 4.145 2.739 0.118 80 442 35 105 1.531 26 9 48 57 -75 1.18786 0 3.43172754 1.7789353
19 5740 16240 100 372 422 172 401 416 123 20 10 0 220 279 182 202 206 41 31 25 0 -1.611 0.273 1.511 1.604 4.569 1.98 0.107 80 460 -11 98 Error -29 18 21 77 -75 1.18786 0 -1.91364246
20 5910 16240 100 456 452 154 388 398 112 36 12 0 217 285 193 198 201 37 38 26 0 3.579 0.736 2.462 2.219 4.823 1.517 0.1 80 432 87 151 1.84 68 19 64 87 -75 1.18786 0 4.25135094 2.0879213

Total number of rows: 20000

Table truncated, full table size 3899 Kbytes.




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