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Sample GSM4612 Query DataSets for GSM4612
Status Public on Jul 25, 2003
Title MGRef Cy3 vs 10APFa Cy5 630590GEO
Sample type RNA
 
Channel 1
Source name 635nm (Cy5) channel, midgut at time point 10 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 25, 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 16520 45150 90 1488 1593 701 199 251 389 96 78 0 1874 1880 924 175 213 143 100 98 0 0.759 0.818 0.767 0.848 1.725 0.718 0.673 52 335 2988 3099 -0.398 1289 1699 1394 1705 -50 1.20661 0 0.91581699 -0.1268693
2 9900 22560 90 434 457 161 144 155 68 96 92 0 599 615 168 156 174 188 98 61 0 0.655 0.682 0.696 0.665 1.581 0.435 0.494 52 386 733 772 -0.611 290 443 313 459 -50 1.20661 0 0.79032955 -0.3394743
3 3110 22640 110 5063 4903 1439 156 177 96 100 100 0 10291 10313 2789 170 196 114 100 100 0 0.485 0.468 0.462 0.463 1.179 0.468 0.944 80 525 15028 14890 -1.044 4907 10121 4747 10143 -50 1.20661 0 0.58520585 -0.7729843
4 10990 27510 110 3902 4120 1981 164 187 173 100 98 0 5614 6366 3008 162 185 108 100 100 0 0.686 0.638 0.633 0.615 1.407 0.635 0.937 80 446 9190 10160 -0.545 3738 5452 3956 6204 -50 1.20661 0 0.82773446 -0.2727603
5 13560 22990 110 709 692 301 170 197 165 88 73 0 695 754 342 168 192 184 91 70 0 1.023 0.891 0.952 0.945 1.904 0.837 0.641 80 500 1066 1108 0.032 539 527 522 586 -50 1.20661 0 1.23436203 0.3037663
6 13000 36470 110 2435 2848 1750 177 245 417 95 88 0 1336 1549 856 160 193 215 100 92 0 1.92 1.923 2.03 1.862 2.253 2.174 0.772 80 467 3434 4060 0.941 2258 1176 2671 1389 -50 1.20661 0 2.3166912 1.2120663
7 16760 49980 110 3715 3719 1100 293 343 272 98 98 0 4020 4197 890 222 282 194 100 100 0 0.901 0.862 0.872 0.808 1.551 0.95 0.881 80 457 7220 7401 -0.15 3422 3798 3426 3975 -50 1.20661 0 1.08715561 0.1205583
8 17660 40620 90 1294 1293 309 183 207 150 100 100 0 1336 1333 299 168 197 149 100 100 0 0.951 0.953 0.925 0.944 1.258 0.937 0.849 52 371 2279 2275 -0.072 1111 1168 1110 1165 -50 1.20661 0 1.14748611 0.1984773
9 15650 49450 60 1564 1779 1165 284 300 127 93 90 0 996 1307 960 224 242 117 84 75 0 1.658 1.38 1.658 1.436 2.247 1.332 0.863 32 176 2052 2578 0.729 1280 772 1495 1083 -50 1.20661 0 2.00055938 1.0004033
10 3110 31570 90 1789 1781 507 146 160 74 100 100 0 3612 3673 907 164 189 117 100 100 0 0.477 0.466 0.456 0.454 1.342 0.452 0.848 52 389 5091 5144 -1.069 1643 3448 1635 3509 -50 1.20661 0 0.57555297 -0.7969793
11 13860 27530 90 967 969 290 162 178 159 100 98 0 1329 1344 374 169 179 62 100 100 0 0.694 0.687 0.682 0.678 1.35 0.689 0.847 52 378 1965 1982 -0.527 805 1160 807 1175 -50 1.20661 0 0.83738734 -0.2560333
12 18160 45160 80 722 777 362 184 193 79 98 92 0 777 833 441 170 180 70 90 90 0 0.886 0.894 0.893 0.941 1.663 0.866 0.812 52 315 1145 1256 -0.174 538 607 593 663 -50 1.20661 0 1.06905646 0.0963383
13 10860 22990 110 1194 1255 388 147 168 135 100 100 0 1435 1486 443 163 176 108 100 100 0 0.823 0.837 0.819 0.834 1.371 0.771 0.733 80 503 2319 2431 -0.281 1047 1272 1108 1323 -50 1.20661 0 0.99304003 -0.0100763
14 15020 36480 90 817 832 195 174 186 74 100 100 0 951 967 251 163 182 132 100 100 0 0.816 0.818 0.806 0.82 1.313 0.803 0.746 52 352 1431 1462 -0.293 643 788 658 804 -50 1.20661 0 0.98459376 -0.0223993
15 16810 27540 120 1013 1158 625 159 168 72 100 98 0 361 405 201 149 162 72 81 62 0 4.028 3.902 3.978 4.381 1.798 3.79 0.757 120 541 1066 1255 2.01 854 212 999 256 -50 1.20661 0 4.86022508 2.2810233
16 14250 36490 90 2647 2581 1111 166 204 191 96 92 0 3731 3535 1550 167 211 250 96 94 0 0.696 0.717 0.717 0.713 2.33 0.681 0.822 52 365 6045 5783 -0.523 2481 3564 2415 3368 -50 1.20661 0 0.83980056 -0.2518813
17 17540 18560 110 1985 2192 787 190 202 86 100 100 0 5472 5910 2226 168 179 82 100 100 0 0.338 0.349 0.352 0.348 1.223 0.332 0.924 80 478 7099 7744 -1.563 1795 5304 2002 5742 -50 1.20661 0 0.40783418 -1.2939453
18 9720 27150 100 3135 3544 1837 144 153 69 100 100 0 4379 5128 2835 161 175 86 100 100 0 0.709 0.685 0.702 0.691 1.581 0.654 0.882 80 460 7209 8367 -0.496 2991 4218 3400 4967 -50 1.20661 0 0.85548649 -0.2251833
19 2390 36280 80 5834 6107 2216 153 178 102 100 100 0 4430 4852 2055 168 197 113 100 100 0 1.333 1.271 1.259 1.293 1.492 1.286 0.857 52 327 9943 10638 0.415 5681 4262 5954 4684 -50 1.20661 0 1.60841113 0.6856363
20 12780 27330 100 2070 2063 725 164 175 104 98 98 0 4084 4054 1115 166 173 65 100 100 0 0.486 0.488 0.498 0.455 1.403 0.503 0.926 80 430 5824 5787 -1.04 1906 3918 1899 3888 -50 1.20661 0 0.58641246 -0.7700123

Total number of rows: 20000

Table truncated, full table size 3857 Kbytes.




Supplementary data files not provided
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