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Sample GSM4592 Query DataSets for GSM4592
Status Public on Jul 25, 2003
Title MGRef Cy3 vs PFa Cy5 710580GEO
Sample type RNA
 
Channel 1
Source name 635nm (Cy5) channel, midgut at puparium formation
Organism Drosophila melanogaster
Extracted molecule total RNA
 
Channel 2
Source name 532nm (Cy3) channel, reference sample
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 the midgut at time points 18 hr. BPF, 4 hr. BPF, PF, 2 hr. APF, 3 hr. APF, 4 hr. APF, 5 hr. APF, 6 hr. APF, 8 hr. APF, 10 hr. APF and 12 hr. APF., vs. a reference sample. Reference sample is a mixture of the midgut samples at time points 18 hr. BPF, 4 hr. BPF, PF, 2 hr. APF, 3 hr. APF, 4 hr. APF, 5 hr. APF, 6 hr. APF, 8 hr. APF, and 12 hr. APF (BPF: before puparium formation; PF: puparium formation, also known as WPP (white prepupa); APF: after puparium formation). Animal staging is 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)). The reference sample is a mixture of the above-described samples. 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 the midgut at one stage 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 at different times in development in the midgut.
· 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 a mixture of the samples from different stages.
· 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 stage.
· 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 TriZOL. 2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol. RNA extraction and amplification were 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 at 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 at 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, its type (e.g., cDNA or oligonucleotide) should be given, along with information that characterizes the reporter molecule unambiguously, in the form of appropriate database reference(s) and sequence (if available).
The FBGN and CG numbers, primer pairs, sequences, and KP number (to tie them to the spots on the array) are all listed in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
· For commercial arrays: a reference to the manufacturer should be provided, including a catalogue number and references to the manufacturer's website if available.
· For non-commercial arrays, the following details should be provided:
o The source of the reporter molecules: for example, the cDNA or oligo collection used, with references.
o The method of reporter preparation.
o The spotting protocols used, including the array substrate, the spotting buffer, and any post-printing processing, including cross-linking.
o Any additional treatment performed prior to hybridization.
We followed a sequence of decreasingly stringent tests to pick optimal primer pairs for the PCR fragments on the arrays. For each gene, we looked for (first) a 500b to 2kb sequence at the 3' end and, failing this, (second) two 200b to 500b sequences in:(1) coding sequence, completely within an EST clot, and unduplicated (BLAST<=10^-4); (2) coding sequence, partially within an EST clot, unduplicated; (3) coding sequence, unduplicated; (4) exon, completely within an EST clot, unduplicated; (5) exon, partially within an EST clot, unduplicated; (6) exon, unduplicated; (7) sequence less than 2kb at the 3' end. See above for the other details about making these arrays.
Sample hybridized to platform version GEO_Dmel200102arrayplatform
Keywords = Drosophila, ecdysone, network, genomic, microarray, organogenesis, EcR, midgut, central nervous system, salivary gland, epidermis, imaginal disc, development
 
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)
GSE543 Midgut metamorphosis timecourse

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 16760 45220 90 1469 1662 997 253 285 207 88 86 0 1366 1484 657 308 324 97 100 100 0 1.149 1.198 1.104 0.968 2.793 1.287 0.604 52 357 2274 2585 0.201 1216 1058 1409 1176 -50 1.34043 0 1.54015407 0.6230753
2 10140 22630 90 520 561 241 235 242 108 80 67 0 528 557 140 261 273 64 100 90 0 1.067 1.101 1.077 0.977 2.18 1.495 0.351 52 378 552 622 0.094 285 267 326 296 -50 1.34043 0 1.43023881 0.5162563
3 3350 22710 120 4726 4857 2513 247 280 173 99 97 0 3380 3416 1543 282 300 159 99 97 0 1.446 1.471 1.482 1.383 1.819 1.534 0.911 120 586 7577 7744 0.532 4479 3098 4610 3134 -50 1.34043 0 1.93826178 0.9547633
4 11230 27590 110 3969 4555 2432 228 249 125 100 97 0 3189 3364 1829 282 296 92 98 97 0 1.287 1.404 1.364 1.444 1.655 1.397 0.928 80 471 6648 7409 0.364 3741 2907 4327 3082 -50 1.34043 0 1.72513341 0.7867083
5 13810 23060 90 626 635 256 229 243 115 92 71 0 706 778 247 277 294 102 100 88 0 0.925 0.81 0.755 0.756 2.145 0.869 0.432 52 392 826 907 -0.112 397 429 406 501 -50 1.34043 0 1.23989775 0.3102213
6 13240 36550 110 491 522 197 246 275 146 72 40 0 1489 1608 610 299 329 216 100 100 0 0.206 0.211 0.204 0.209 1.767 0.188 0.417 80 471 1435 1585 -2.28 245 1190 276 1309 -50 1.34043 0 0.27612858 -1.8565883
7 16990 50060 110 1437 1430 340 253 277 139 100 100 0 1898 1921 377 315 352 149 100 100 0 0.748 0.733 0.717 0.72 1.325 0.721 0.776 80 460 2767 2783 -0.419 1184 1583 1177 1606 -50 1.34043 0 1.00264164 0.0038063
8 17890 40710 100 1186 1214 385 250 269 125 100 98 0 1191 1206 341 304 310 74 100 98 0 1.055 1.069 1.05 1.065 1.677 1.102 0.755 80 412 1823 1866 0.078 936 887 964 902 -50 1.34043 0 1.41415365 0.4999393
9 15900 49510 70 519 564 307 249 257 108 75 59 0 881 1047 650 299 306 67 93 87 0 0.464 0.421 0.41 0.416 3.02 0.411 0.58 32 236 852 1063 -1.108 270 582 315 748 -50 1.34043 0 0.62195952 -0.6851073
10 3350 31630 90 2618 2426 871 259 303 219 100 100 0 2540 2485 748 281 299 116 100 100 0 1.044 0.983 0.944 0.95 1.627 1.034 0.78 52 379 4618 4371 0.062 2359 2259 2167 2204 -50 1.34043 0 1.39940892 0.4848183
11 14100 27600 90 643 635 237 218 236 102 88 80 0 1052 1032 229 281 282 62 100 100 0 0.551 0.555 0.584 0.486 2.03 0.563 0.537 52 370 1196 1168 -0.859 425 771 417 751 -50 1.34043 0 0.73857693 -0.4371803
12 18390 45220 90 582 641 318 269 277 120 75 61 0 718 821 358 303 317 95 92 82 0 0.754 0.718 0.8 0.783 2.258 0.875 0.499 52 386 728 890 -0.407 313 415 372 518 -50 1.34043 0 1.01068422 0.0153323
13 11100 23060 100 1044 1076 349 236 250 151 97 92 0 1294 1301 255 273 289 85 100 100 0 0.791 0.817 0.875 0.796 1.477 0.876 0.668 80 441 1829 1868 -0.338 808 1021 840 1028 -50 1.34043 0 1.06028013 0.0844453
14 15270 36550 100 553 564 188 237 256 146 81 53 0 791 802 149 296 301 82 100 100 0 0.638 0.646 0.653 0.585 1.972 0.691 0.477 80 424 811 833 -0.648 316 495 327 506 -50 1.34043 0 0.85519434 -0.2256763
15 17050 27610 90 374 397 143 230 234 94 69 38 0 585 595 113 281 282 62 100 98 0 0.474 0.532 0.469 0.504 2.266 0.7 0.164 52 388 448 481 -1.078 144 304 167 314 -50 1.34043 0 0.63536382 -0.6543453
16 14480 36550 80 2555 2382 949 240 284 296 92 88 0 2962 2835 1045 307 356 354 96 92 0 0.872 0.847 0.884 0.858 2.115 0.851 0.823 52 306 4970 4670 -0.198 2315 2655 2142 2528 -50 1.34043 0 1.16885496 0.2250963
17 17790 18630 100 1816 1951 732 250 261 139 100 100 0 3472 3740 1171 269 274 62 100 100 0 0.489 0.49 0.482 0.472 1.335 0.493 0.862 80 454 4769 5172 -1.032 1566 3203 1701 3471 -50 1.34043 0 0.65547027 -0.6093983
18 9960 27210 100 2627 3160 1995 233 249 120 97 97 0 3147 3557 1794 276 298 144 100 100 0 0.834 0.892 0.825 0.777 1.776 0.958 0.844 80 442 5265 6208 -0.262 2394 2871 2927 3281 -50 1.34043 0 1.11791862 0.1608153
19 2640 36350 90 956 1278 812 250 286 201 86 69 0 2706 2879 1596 284 301 103 100 98 0 0.291 0.396 0.406 0.333 1.765 0.408 0.76 52 388 3128 3623 -1.778 706 2422 1028 2595 -50 1.34043 0 0.39006513 -1.3582133
20 13020 27410 100 2409 2425 770 237 245 98 100 100 0 2980 2795 797 285 304 157 100 100 0 0.806 0.872 0.865 0.857 1.403 0.886 0.889 80 442 4867 4698 -0.311 2172 2695 2188 2510 -50 1.34043 0 1.08038658 0.1115483

Total number of rows: 20000

Table truncated, full table size 3795 Kbytes.




Supplementary data files not provided
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