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Sample GSM4588 Query DataSets for GSM4588
Status Public on Jul 25, 2003
Title MGRef Cy3 vs 4BPFa Cy5 710570GEO
Sample type RNA
 
Channel 1
Source name 635nm (Cy5) channel, midgut at time point 4 hr. before 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 16630 45380 100 1833 2035 1177 260 292 233 96 90 0 1501 1708 878 334 343 87 100 100 0 1.348 1.292 1.3 1.247 1.862 1.357 0.727 80 411 2740 3149 0.431 1573 1167 1775 1374 -50 1.39332 0 1.87819536 0.9093473
2 10010 22770 90 459 495 184 212 232 117 84 59 0 632 657 150 278 283 65 100 98 0 0.698 0.747 0.766 0.733 1.871 0.938 0.382 52 379 601 662 -0.519 247 354 283 379 -50 1.39332 0 0.97253736 -0.0401743
3 3210 22860 120 3950 4290 2490 239 275 269 95 90 0 3531 3722 1583 312 337 168 100 99 0 1.153 1.188 1.156 0.993 2.126 1.308 0.87 120 522 6930 7461 0.205 3711 3219 4051 3410 -50 1.39332 0 1.60649796 0.6839193
4 11090 27740 110 4088 4249 1940 235 254 135 100 100 0 4774 4856 2264 310 332 137 100 97 0 0.863 0.883 0.874 0.91 1.595 0.872 0.923 80 446 8317 8560 -0.212 3853 4464 4014 4546 -50 1.39332 0 1.20243516 0.2659593
5 13670 23220 110 568 584 241 219 233 162 78 55 0 742 784 249 272 287 78 98 96 0 0.743 0.713 0.788 0.657 2.53 0.964 0.293 80 546 819 877 -0.429 349 470 365 512 -50 1.39332 0 1.03523676 0.0499613
6 13090 36700 100 488 534 241 218 247 181 71 33 0 1658 1918 837 308 334 181 100 100 0 0.2 0.196 0.209 0.176 2.227 0.183 0.544 80 436 1620 1926 -2.322 270 1350 316 1610 -50 1.39332 0 0.278664 -1.8434013
7 16850 50210 120 2284 2099 704 337 370 194 97 93 0 2722 2661 902 367 426 263 96 94 0 0.827 0.768 0.763 0.773 1.983 0.752 0.818 120 471 4302 4056 -0.274 1947 2355 1762 2294 -50 1.39332 0 1.15227564 0.2044863
8 17740 40850 90 1075 1138 339 227 256 150 100 100 0 1160 1200 235 283 327 338 100 90 0 0.967 0.993 0.923 0.958 1.444 1.044 0.628 52 357 1725 1828 -0.049 848 877 911 917 -50 1.39332 0 1.34734044 0.4301143
9 15750 49660 50 802 983 458 357 358 131 83 75 0 2057 2050 596 406 408 79 100 100 0 0.27 0.381 0.321 0.295 1.934 0.425 0.636 12 104 2096 2270 -1.891 445 1651 626 1644 -50 1.39332 0 0.3761964 -1.4104423
10 3210 31790 90 2783 2693 975 262 287 165 100 100 0 2267 2143 683 316 355 256 100 96 0 1.292 1.331 1.253 1.309 1.575 1.392 0.791 52 388 4472 4258 0.37 2521 1951 2431 1827 -50 1.39332 0 1.80016944 0.8481333
11 13960 27750 100 613 649 275 229 245 115 91 70 0 1233 1241 429 303 311 100 100 97 0 0.413 0.448 0.441 0.426 1.785 0.44 0.578 80 440 1314 1358 -1.276 384 930 420 938 -50 1.39332 0 0.57544116 -0.7972603
12 18250 45380 70 721 742 267 256 277 124 93 81 0 887 991 329 344 353 95 100 96 0 0.856 0.751 0.761 0.705 1.9 0.716 0.435 32 236 1008 1133 -0.224 465 543 486 647 -50 1.39332 0 1.19268192 0.2542093
13 10960 23210 110 888 896 360 210 228 171 91 83 0 1429 1479 336 273 288 126 100 100 0 0.587 0.569 0.557 0.524 1.779 0.619 0.563 80 483 1834 1892 -0.77 678 1156 686 1206 -50 1.39332 0 0.81787884 -0.2900413
14 15120 36700 90 591 627 225 217 235 127 96 71 0 817 797 153 298 324 194 98 75 0 0.721 0.822 0.748 0.749 1.891 0.893 0.393 52 384 893 909 -0.473 374 519 410 499 -50 1.39332 0 1.00458372 0.0065983
15 16910 27770 90 363 378 143 229 249 120 51 23 0 639 646 150 283 301 124 96 78 0 0.376 0.41 0.478 0.426 2.714 0.448 0.118 52 379 490 512 -1.41 134 356 149 363 -50 1.39332 0 0.52388832 -0.9326693
16 14360 36710 100 2093 1938 1079 233 258 169 93 81 0 2889 2466 1348 300 313 94 97 91 0 0.718 0.787 0.804 0.835 2.351 0.775 0.765 80 385 4449 3871 -0.477 1860 2589 1705 2166 -50 1.39332 0 1.00040376 0.0005823
17 17650 18780 110 6813 7090 3104 228 252 157 100 100 0 3703 3790 1123 270 273 69 100 100 0 1.918 1.949 1.861 1.839 1.379 2.128 0.898 80 488 10018 10382 0.94 6585 3433 6862 3520 -50 1.39332 0 2.67238776 1.4181293
18 9830 27360 110 2564 3008 1404 221 233 103 100 100 0 3970 4704 1977 303 309 71 100 100 0 0.639 0.633 0.608 0.616 1.344 0.629 0.866 80 516 6010 7188 -0.646 2343 3667 2787 4401 -50 1.39332 0 0.89033148 -0.1675863
19 2490 36490 90 1404 1389 786 283 303 142 90 80 0 3436 3376 1480 389 413 131 100 100 0 0.368 0.37 0.333 0.314 2.165 0.378 0.718 52 388 4168 4093 -1.443 1121 3047 1106 2987 -50 1.39332 0 0.51274176 -0.9636963
20 12880 27560 100 3402 3154 1090 227 244 114 98 97 0 3798 3776 1050 306 321 146 100 100 0 0.909 0.844 0.841 0.779 1.654 0.854 0.912 80 442 6667 6397 -0.137 3175 3492 2927 3470 -50 1.39332 0 1.26652788 0.3408793

Total number of rows: 20000

Table truncated, full table size 3799 Kbytes.




Supplementary data files not provided
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