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Sample GSM4594 Query DataSets for GSM4594
Status Public on Jul 25, 2003
Title MGRef Cy3 vs 2APFa Cy5 670580GEO
Sample type RNA
 
Channel 1
Source name 635nm (Cy5) channel, midgut at time point 2 hr. after 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 16430 45200 90 1338 1620 1091 154 181 151 98 90 0 1319 1530 915 258 278 105 92 86 0 1.116 1.153 1.096 1.22 2.455 1.245 0.631 52 354 2245 2738 0.158 1184 1061 1466 1272 -50 1.17866 0 1.31538456 0.3954853
2 9820 22600 90 394 414 122 145 152 61 96 88 0 727 703 135 236 241 61 100 100 0 0.507 0.576 0.552 0.525 1.813 0.562 0.553 52 386 740 736 -0.98 249 491 269 467 -50 1.17866 0 0.59758062 -0.7427953
3 3020 22690 120 6128 6023 2734 178 198 130 100 99 0 5402 5502 2586 225 244 112 100 99 0 1.149 1.108 1.109 1.133 1.659 1.099 0.931 120 551 11127 11122 0.201 5950 5177 5845 5277 -50 1.17866 0 1.35428034 0.4375263
4 10890 27560 110 6862 6760 2953 158 183 135 100 100 0 6409 6241 2975 271 291 139 100 100 0 1.092 1.106 1.092 1.198 1.585 1.076 0.944 80 459 12842 12572 0.127 6704 6138 6602 5970 -50 1.17866 0 1.28709672 0.3641203
5 13470 23040 100 649 670 282 147 166 109 96 82 0 949 1000 360 250 265 103 100 98 0 0.718 0.697 0.755 0.685 1.734 0.644 0.676 80 460 1201 1273 -0.478 502 699 523 750 -50 1.17866 0 0.84627788 -0.2407973
6 12900 36520 100 657 837 454 138 151 89 100 95 0 1728 2170 1148 254 272 210 100 100 0 0.352 0.365 0.381 0.351 1.704 0.343 0.748 80 430 1993 2615 -1.506 519 1474 699 1916 -50 1.17866 0 0.41488832 -1.2692053
7 16650 50030 110 1693 1654 429 164 186 121 100 98 0 1763 1688 308 222 250 123 100 100 0 0.992 1.016 1.026 0.982 1.547 1.05 0.783 80 482 3070 2956 -0.011 1529 1541 1490 1466 -50 1.17866 0 1.16923072 0.2255603
8 17550 40670 90 1121 1115 344 167 183 108 98 98 0 1684 1665 397 261 283 119 100 100 0 0.67 0.675 0.685 0.672 1.462 0.678 0.793 52 366 2377 2352 -0.577 954 1423 948 1404 -50 1.17866 0 0.7897022 -0.3406193
9 15570 49490 60 633 665 409 160 180 111 84 68 0 814 817 400 234 247 70 93 87 0 0.816 0.866 0.794 0.892 2.142 0.93 0.591 32 176 1053 1088 -0.294 473 580 505 583 -50 1.17866 0 0.96178656 -0.0562113
10 3020 31610 90 3774 3651 1034 156 175 113 100 100 0 4021 3848 872 238 250 92 100 100 0 0.956 0.968 0.966 0.946 1.367 1.018 0.871 52 362 7401 7105 -0.064 3618 3783 3495 3610 -50 1.17866 0 1.12679896 0.1722303
11 13770 27570 100 1269 1260 632 159 174 211 90 85 0 1793 1760 759 261 267 88 97 96 0 0.725 0.734 0.748 0.725 1.535 0.78 0.732 80 442 2642 2600 -0.465 1110 1532 1101 1499 -50 1.17866 0 0.8545285 -0.2267993
12 18070 45190 80 616 757 450 166 173 72 92 78 0 964 957 424 255 260 56 98 94 0 0.635 0.842 0.813 0.754 2.238 0.9 0.727 52 328 1159 1293 -0.656 450 709 591 702 -50 1.17866 0 0.7484491 -0.4180243
13 10770 23030 110 1143 1132 376 153 159 66 100 100 0 1657 1749 396 236 246 109 100 100 0 0.697 0.647 0.644 0.601 1.614 0.658 0.769 80 496 2411 2492 -0.521 990 1421 979 1513 -50 1.17866 0 0.82152602 -0.2836223
14 14930 36520 90 611 616 219 135 146 64 98 98 0 1013 1033 233 248 254 60 100 100 0 0.622 0.613 0.59 0.561 1.763 0.624 0.691 52 352 1241 1266 -0.684 476 765 481 785 -50 1.17866 0 0.73312652 -0.4478663
15 16730 27580 90 750 707 307 165 180 83 94 82 0 1226 1148 292 269 281 102 100 98 0 0.611 0.617 0.608 0.504 2.087 0.648 0.748 52 365 1542 1421 -0.71 585 957 542 879 -50 1.17866 0 0.72016126 -0.4736083
16 14150 36530 80 2524 2309 927 136 160 131 98 96 0 3427 3382 1247 257 295 214 98 98 0 0.753 0.695 0.714 0.653 2.038 0.663 0.835 52 299 5558 5298 -0.409 2388 3170 2173 3125 -50 1.17866 0 0.88753098 -0.1721313
17 17460 18600 110 1686 1891 729 194 210 97 100 100 0 6173 6575 2324 271 284 86 100 100 0 0.253 0.269 0.269 0.263 1.345 0.263 0.889 80 478 7394 8001 -1.984 1492 5902 1697 6304 -50 1.17866 0 0.29820098 -1.7456433
18 9630 27190 100 3980 4593 2880 159 166 67 100 100 0 5567 6366 4036 262 267 71 98 98 0 0.72 0.726 0.738 0.758 1.795 0.71 0.902 80 410 9126 10538 -0.473 3821 5305 4434 6104 -50 1.17866 0 0.8486352 -0.2367843
19 2300 36330 90 1815 2412 1444 181 209 162 100 98 0 2508 3244 1880 235 253 94 100 100 0 0.719 0.741 0.725 0.726 1.758 0.73 0.763 52 394 3907 5240 -0.476 1634 2273 2231 3009 -50 1.17866 0 0.84745654 -0.2387893
20 12690 27380 100 4357 4088 1468 163 193 266 97 97 0 5068 4965 1566 271 283 98 100 100 0 0.874 0.836 0.843 0.79 1.453 0.85 0.915 80 442 8991 8619 -0.194 4194 4797 3925 4694 -50 1.17866 0 1.03014884 0.0428533

Total number of rows: 20000

Table truncated, full table size 3829 Kbytes.




Supplementary data files not provided
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