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Sample GSM4508 Query DataSets for GSM4508
Status Public on Jul 25, 2003
Title wholeRefCy3 vs SG1Cy5 710520GEO
Sample type RNA
 
Channel 1
Source name Salivary gland 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 3090 15250 100 294 368 234 253 265 112 37 23 0 371 487 267 292 300 69 52 40 0 0.519 0.59 0.891 1.076 4.597 0.926 0.217 80 486 120 310 -0.946 41 79 115 195 -75 1.2877 0 0.6683163 -0.5813973
2 3260 15250 100 315 353 169 251 270 118 38 18 0 354 414 198 301 315 81 36 25 0 1.208 0.903 1.218 1.458 3.322 1.126 0.204 80 442 117 215 0.272 64 53 102 113 -75 1.2877 0 1.5555416 0.6374173
3 3430 15250 100 351 392 228 260 276 115 40 21 0 346 471 347 303 315 80 36 23 0 2.116 0.786 0.926 0.975 3.367 0.661 0.287 80 442 134 300 1.082 91 43 132 168 -75 1.2877 0 2.7247732 1.4461363
4 3600 15250 100 287 326 193 268 286 125 31 13 0 340 399 190 327 343 100 30 15 0 1.462 0.806 1.489 1.323 2.777 1.017 0.144 80 442 32 130 0.547 19 13 58 72 -75 1.2877 0 1.8826174 0.9127403
5 3770 15250 100 343 404 231 277 291 124 38 22 0 372 541 378 343 365 116 32 25 0 2.276 0.641 0.69 0.982 3.535 0.675 0.351 80 420 95 325 1.186 66 29 127 198 -75 1.2877 0 2.9308052 1.5512973
6 3940 15250 100 342 395 225 309 323 132 22 13 0 398 505 294 359 387 128 32 17 0 0.846 0.589 0.973 0.978 4.741 0.841 0.324 80 442 72 232 -0.241 33 39 86 146 -75 1.2877 0 1.0893942 0.1235263
7 4110 15260 100 331 341 173 310 315 131 21 8 0 346 409 198 344 371 128 17 12 0 10.5 0.477 1.319 1.662 3.507 0.914 0.295 80 450 23 96 3.392 21 2 31 65 -75 1.2877 0 13.52085 3.7571143
8 4280 15260 100 277 333 189 315 332 145 17 10 0 341 425 229 354 385 131 20 13 0 2.923 0.254 1.597 1.894 4.402 1.031 0.194 80 450 -51 89 1.547 -38 -13 18 71 -75 1.2877 0 3.7639471 1.9122463
9 4450 15260 100 300 355 215 342 351 142 21 10 0 358 448 224 387 396 97 21 15 0 1.448 0.213 1.228 1.255 3.111 1.514 0.143 80 442 -71 74 0.534 -42 -29 13 61 -75 1.2877 0 1.8645896 0.8988583
10 4620 15260 100 345 389 220 360 384 153 22 11 0 383 443 195 403 414 96 20 16 0 0.75 0.725 1.079 1.172 3.234 2.037 0.175 80 442 -35 69 -0.415 -15 -20 29 40 -75 1.2877 0 0.965775 -0.0502413
11 4790 15260 100 427 468 234 448 472 187 15 7 0 434 526 294 467 487 142 16 13 0 0.636 0.339 1.281 1.326 3.259 1.112 0.143 80 442 -54 79 -0.652 -21 -33 20 59 -75 1.2877 0 0.8189772 -0.2881053
12 4960 15260 100 460 525 269 432 467 218 23 10 0 454 529 231 442 466 143 22 18 0 2.333 1.069 1.103 1.091 3.945 2.309 0.12 80 453 40 180 1.222 28 12 93 87 -75 1.2877 0 3.0042041 1.5869833
13 5130 15260 100 475 554 324 430 457 225 23 12 0 418 503 259 402 429 144 23 15 0 2.813 1.228 1.381 1.45 3.193 1.541 0.312 80 442 61 225 1.492 45 16 124 101 -75 1.2877 0 3.6223001 1.8569063
14 5300 15260 100 422 587 709 369 403 210 23 13 0 386 533 341 348 369 140 33 23 0 1.395 1.178 1.423 1.396 3.48 3.134 0.237 80 442 91 403 0.48 53 38 218 185 -75 1.2877 0 1.7963415 0.8450623
15 5470 15270 100 410 493 297 318 381 491 12 2 0 389 524 601 306 335 147 31 21 0 1.108 0.803 1.628 1.557 3.949 4.335 0.065 80 440 175 393 0.149 92 83 175 218 -75 1.2877 0 1.4267716 0.5127543
16 5640 15270 100 272 428 392 295 324 190 22 17 0 319 435 370 296 327 153 18 13 0 -1 0.957 1.352 1.592 3.455 1.665 0.162 80 440 0 272 Error -23 23 133 139 -75 1.2877 0 -1.2877
17 5810 15270 100 276 338 216 253 275 133 23 13 0 316 400 274 280 296 104 23 16 0 0.639 0.708 1.25 1.098 3.748 0.803 0.113 80 450 59 205 -0.646 23 36 85 120 -75 1.2877 0 0.8228403 -0.2813163
18 5980 15270 100 266 371 370 242 271 142 21 15 0 272 350 242 269 295 115 18 13 0 8 1.593 1.21 1.26 4.525 2.455 0.142 80 442 27 210 3 24 3 129 81 -75 1.2877 0 10.3016 3.3647973
19 6150 15270 100 259 377 309 250 271 140 32 22 0 287 385 285 265 287 106 28 16 0 0.409 1.058 1.794 1.285 4.367 0.984 0.105 80 452 31 247 -1.29 9 22 127 120 -75 1.2877 0 0.5266693 -0.9250313
20 6320 15270 100 324 388 370 247 267 141 31 15 0 322 423 273 265 281 78 41 28 0 1.351 0.892 1.637 1.55 4.255 2.084 0.166 80 442 134 299 0.434 77 57 141 158 -75 1.2877 0 1.7396827 0.7988243

Total number of rows: 20000

Table truncated, full table size 3962 Kbytes.




Supplementary data files not provided
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