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Sample GSM4615 Query DataSets for GSM4615
Status Public on Jul 25, 2003
Title MGRef Cy3 vs 12APFa Cy5 680590GEO
Sample type RNA
 
Channel 1
Source name 635nm (Cy5) channel, midgut at time point 12 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 16840 45150 90 1619 1784 866 226 260 143 100 96 0 2523 2617 1301 213 241 148 100 100 0 0.603 0.648 0.675 0.639 1.984 0.561 0.638 52 363 3703 3962 -0.73 1393 2310 1558 2404 -50 1.31168 0 0.79094304 -0.3383543
2 10220 22550 80 419 419 131 197 208 90 90 55 0 487 512 196 183 193 68 98 90 0 0.73 0.675 0.83 0.618 2.515 0.578 0.307 52 340 526 551 -0.454 222 304 222 329 -50 1.31168 0 0.9575264 -0.0626163
3 3430 22640 120 3834 3844 1882 217 242 163 99 99 0 5638 5868 2705 187 210 117 100 100 0 0.664 0.638 0.635 0.611 1.657 0.64 0.924 120 534 9068 9308 -0.592 3617 5451 3627 5681 -50 1.31168 0 0.87095552 -0.1993293
4 11300 27510 100 4297 4545 2176 204 234 137 97 97 0 5208 5645 2730 192 229 154 100 100 0 0.816 0.796 0.813 0.748 1.772 0.787 0.925 80 450 9109 9794 -0.293 4093 5016 4341 5453 -50 1.31168 0 1.07033088 0.0980573
5 13870 23000 110 649 672 252 196 207 93 96 88 0 690 707 261 179 188 65 100 97 0 0.886 0.902 0.868 0.897 2.144 0.829 0.506 80 514 964 1004 -0.174 453 511 476 528 -50 1.31168 0 1.16214848 0.2167943
6 13300 36470 110 1669 2007 1046 218 251 196 98 97 0 2608 3040 1268 201 222 106 100 100 0 0.603 0.63 0.624 0.591 1.513 0.63 0.76 80 445 3858 4628 -0.73 1451 2407 1789 2839 -50 1.31168 0 0.79094304 -0.3383543
7 17060 49980 120 1824 1782 744 247 277 163 95 89 0 2644 2424 1097 227 284 234 92 85 0 0.652 0.699 0.681 0.698 2.238 0.668 0.814 120 514 3994 3732 -0.616 1577 2417 1535 2197 -50 1.31168 0 0.85521536 -0.2256403
8 17960 40630 100 1512 1588 479 208 224 101 100 100 0 1494 1513 456 200 223 108 100 100 0 1.008 1.051 1.068 1.055 1.332 1.048 0.89 80 440 2598 2693 0.011 1304 1294 1380 1313 -50 1.31168 0 1.32217344 0.4029113
9 15970 49430 100 354 524 423 236 256 127 46 36 0 367 578 483 226 251 130 51 36 0 0.837 0.818 1.162 0.975 3.903 0.776 0.438 80 455 259 640 -0.257 118 141 288 352 -50 1.31168 0 1.09787616 0.1347153
10 3420 31560 90 2225 2260 621 240 275 187 100 100 0 3599 3516 969 216 252 263 100 100 0 0.587 0.612 0.611 0.609 1.351 0.599 0.851 52 366 5368 5320 -0.769 1985 3383 2020 3300 -50 1.31168 0 0.76995616 -0.3771523
11 14180 27520 100 866 905 378 205 213 94 96 91 0 1244 1287 449 190 201 72 100 100 0 0.627 0.638 0.61 0.577 1.674 0.667 0.799 80 442 1715 1797 -0.673 661 1054 700 1097 -50 1.31168 0 0.82242336 -0.2820473
12 18470 45150 80 862 1029 744 249 303 321 69 48 0 874 1219 1006 223 242 111 94 75 0 0.942 0.783 0.886 0.802 2.504 0.837 0.412 52 316 1264 1776 -0.087 613 651 780 996 -50 1.31168 0 1.23560256 0.3052153
13 11170 22990 110 1566 1598 447 193 206 111 100 100 0 1467 1451 439 180 188 60 100 98 0 1.067 1.105 1.071 1.144 1.595 1.082 0.805 80 514 2660 2676 0.093 1373 1287 1405 1271 -50 1.31168 0 1.39956256 0.4849763
14 15340 36470 100 2809 2714 787 225 243 129 100 98 0 1143 1127 324 204 227 111 98 96 0 2.752 2.697 2.604 2.766 1.738 2.934 0.752 80 420 3523 3412 1.46 2584 939 2489 923 -50 1.31168 0 3.60974336 1.8518963
15 17140 27530 90 821 762 245 207 221 84 96 92 0 555 577 167 181 193 63 100 96 0 1.642 1.402 1.317 1.298 1.94 1.495 0.647 52 366 988 951 0.715 614 374 555 396 -50 1.31168 0 2.15377856 1.1068703
16 14540 36480 90 3620 3376 1378 233 250 120 98 92 0 6066 5814 1772 206 239 200 100 100 0 0.578 0.56 0.56 0.477 2.29 0.549 0.759 52 354 9247 8751 -0.791 3387 5860 3143 5608 -50 1.31168 0 0.75815104 -0.3994433
17 17870 18550 110 1837 1979 729 209 218 87 100 100 0 5141 5487 1921 199 206 65 100 100 0 0.329 0.335 0.337 0.325 1.318 0.332 0.932 80 466 6570 7058 -1.602 1628 4942 1770 5288 -50 1.31168 0 0.43154272 -1.2124253
18 10030 27130 100 3431 4042 2516 196 219 221 98 95 0 3854 5040 2763 189 213 200 100 100 0 0.883 0.793 0.764 0.706 1.701 0.827 0.868 80 421 6900 8697 -0.18 3235 3665 3846 4851 -50 1.31168 0 1.15821344 0.2119013
19 2700 36270 90 2848 2734 1148 250 304 254 100 98 0 4809 4774 2099 233 275 154 100 100 0 0.568 0.547 0.544 0.542 1.553 0.529 0.798 52 387 7174 7025 -0.817 2598 4576 2484 4541 -50 1.31168 0 0.74503424 -0.4246213
20 13090 27330 100 3267 3090 1098 205 216 87 97 97 0 4315 4185 1271 189 198 66 100 100 0 0.742 0.722 0.73 0.674 1.66 0.733 0.924 80 418 7188 6881 -0.43 3062 4126 2885 3996 -50 1.31168 0 0.97326656 -0.0390933

Total number of rows: 20000

Table truncated, full table size 3840 Kbytes.




Supplementary data files not provided
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