NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM4600 Query DataSets for GSM4600
Status Public on Jul 25, 2003
Title MGRef Cy3 vs 4APFa Cy5 670570GEO
Sample type RNA
 
Channel 1
Source name 635nm (Cy5) channel, midgut at time point 4 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 16570 45390 90 1293 1546 1089 192 257 640 73 40 0 1310 1557 983 250 311 449 86 67 0 1.039 1.036 0.991 1.066 1.748 1.034 0.693 52 381 2161 2661 0.055 1101 1060 1354 1307 -50 1.10211 0 1.14509229 0.1954643
2 9940 22810 90 632 664 189 202 228 128 98 92 0 814 833 233 283 300 107 100 96 0 0.81 0.84 0.823 0.848 1.643 0.797 0.5 52 376 961 1012 -0.304 430 531 462 550 -50 1.10211 0 0.8927091 -0.1637383
3 3160 22880 120 4242 4334 2283 204 232 146 100 97 0 5003 5201 2602 270 295 123 99 98 0 0.853 0.838 0.864 0.836 1.988 0.839 0.908 120 554 8771 9061 -0.229 4038 4733 4130 4931 -50 1.10211 0 0.94009983 -0.0891143
4 11030 27750 100 5705 5786 2930 209 250 217 97 96 0 5967 6105 2625 291 329 257 100 100 0 0.968 0.959 0.923 0.851 1.764 0.999 0.924 80 438 11172 11391 -0.046 5496 5676 5577 5814 -50 1.10211 0 1.06684248 0.0933473
5 13600 23240 110 821 919 380 196 215 102 98 91 0 941 968 373 273 284 99 93 92 0 0.936 1.04 1.012 1.003 2.018 1.015 0.652 80 526 1293 1418 -0.096 625 668 723 695 -50 1.10211 0 1.03157496 0.0448493
6 13050 36710 110 1861 2221 1261 180 247 394 93 91 0 2416 2604 1208 248 303 321 100 97 0 0.775 0.866 0.868 0.784 2.096 0.962 0.837 80 494 3849 4397 -0.367 1681 2168 2041 2356 -50 1.10211 0 0.85413525 -0.2274643
7 16790 50230 100 2980 3051 745 266 320 198 100 100 0 3123 3122 657 304 364 220 100 100 0 0.963 0.988 0.985 0.982 1.28 0.989 0.833 80 418 5533 5603 -0.055 2714 2819 2785 2818 -50 1.10211 0 1.06133193 0.0858763
8 17680 40870 110 1044 1113 382 187 199 91 100 98 0 1534 1496 388 260 289 173 100 100 0 0.673 0.749 0.78 0.713 1.421 0.762 0.782 80 509 2131 2162 -0.572 857 1274 926 1236 -50 1.10211 0 0.74172003 -0.4310533
9 15700 49680 60 1325 2232 1825 239 261 110 93 81 0 1827 2463 1886 304 321 94 96 93 0 0.713 0.923 0.938 0.849 2.247 0.934 0.827 32 176 2609 4152 -0.488 1086 1523 1993 2159 -50 1.10211 0 0.78580443 -0.3477583
10 3150 31810 90 3588 3389 1120 214 235 118 100 100 0 3663 3325 1307 282 308 154 100 100 0 0.998 1.043 1.068 1.097 1.45 0.99 0.882 52 363 6755 6218 -0.003 3374 3381 3175 3043 -50 1.10211 0 1.09990578 0.1373803
11 13900 27770 100 2110 1983 872 190 204 128 97 97 0 1624 1681 633 273 281 67 100 100 0 1.421 1.273 1.29 1.196 1.754 1.313 0.897 80 442 3271 3201 0.507 1920 1351 1793 1408 -50 1.10211 0 1.56609831 0.6471753
12 18200 45390 90 721 899 630 198 219 161 82 73 0 909 1001 525 240 259 112 94 86 0 0.782 0.921 0.859 0.81 1.935 1.03 0.724 52 379 1192 1462 -0.355 523 669 701 761 -50 1.10211 0 0.86185002 -0.2144913
13 10900 23230 100 1746 1710 457 187 202 101 100 100 0 1616 1600 382 264 282 142 100 100 0 1.153 1.14 1.13 1.137 1.416 1.118 0.811 80 464 2911 2859 0.206 1559 1352 1523 1336 -50 1.10211 0 1.27073283 0.3456613
14 15070 36710 80 665 694 194 189 215 206 94 65 0 1137 1158 192 252 268 85 100 100 0 0.538 0.557 0.569 0.517 1.585 0.651 0.457 52 310 1361 1411 -0.895 476 885 505 906 -50 1.10211 0 0.59293518 -0.7540543
15 16860 27780 80 593 605 226 186 194 79 94 84 0 871 851 198 259 265 66 100 100 0 0.665 0.708 0.713 0.619 1.98 0.754 0.647 52 316 1019 1011 -0.589 407 612 419 592 -50 1.10211 0 0.73290315 -0.4483063
16 14280 36720 90 3579 3350 1242 186 229 218 96 92 0 4768 4597 1172 255 295 231 100 98 0 0.752 0.729 0.752 0.648 1.907 0.746 0.78 52 371 7906 7506 -0.412 3393 4513 3164 4342 -50 1.10211 0 0.82878672 -0.2709273
17 17580 18800 110 1759 2083 910 198 217 109 100 100 0 4569 5637 2130 248 260 143 100 100 0 0.361 0.35 0.346 0.336 1.255 0.347 0.9 80 500 5882 7274 -1.469 1561 4321 1885 5389 -50 1.10211 0 0.39786171 -1.3296613
18 9760 27370 90 6231 6292 3287 194 217 129 100 100 0 6655 6750 3240 274 291 95 100 100 0 0.946 0.942 0.915 0.912 1.297 0.98 0.926 52 372 12418 12574 -0.08 6037 6381 6098 6476 -50 1.10211 0 1.04259606 0.0601803
19 2440 36520 80 5742 5683 2370 218 244 129 100 100 0 4206 3878 1716 276 296 115 98 98 0 1.406 1.517 1.505 1.643 1.673 1.528 0.872 52 334 9454 9067 0.491 5524 3930 5465 3602 -50 1.10211 0 1.54956666 0.6318653
20 12820 27580 110 5094 4811 1219 201 219 111 100 100 0 4451 4316 1300 282 294 88 100 100 0 1.174 1.143 1.156 1.181 1.296 1.119 0.941 80 492 9062 8644 0.231 4893 4169 4610 4034 -50 1.10211 0 1.29387714 0.3717013

Total number of rows: 20000

Table truncated, full table size 3889 Kbytes.




Supplementary data files not provided
| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap