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Sample GSM4597 Query DataSets for GSM4597
Status Public on Jul 25, 2003
Title MGRef Cy3 vs 3APFa Cy5 670570GEO
Sample type RNA
 
Channel 1
Source name 635nm (Cy5) channel, midgut at time point 3 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 16530 45210 90 1453 1802 1194 173 211 226 94 86 0 1661 1897 1144 242 264 186 94 90 0 0.902 0.984 1.004 0.945 2.182 1.032 0.736 52 364 2699 3284 -0.149 1280 1419 1629 1655 -50 1.1948 0 1.0777096 0.1079683
2 9880 22620 90 461 476 122 169 184 203 76 23 0 635 647 146 226 232 83 100 96 0 0.714 0.729 0.74 0.718 1.68 0.848 0.495 52 376 701 728 -0.486 292 409 307 421 -50 1.1948 0 0.8530872 -0.2292353
3 3080 22700 110 7621 7392 2024 209 240 171 100 100 0 7476 7386 1481 255 280 182 100 100 0 1.026 1.007 1.01 0.97 1.329 1.055 0.927 80 503 14633 14314 0.038 7412 7221 7183 7131 -50 1.1948 0 1.2258648 0.2938003
4 10970 27580 120 4442 4591 3422 172 179 77 99 96 0 3619 4240 3064 230 241 64 100 99 0 1.26 1.102 1.096 1.072 2.062 1.111 0.936 120 489 7659 8429 0.333 4270 3389 4419 4010 -50 1.1948 0 1.505448 0.5901933
5 13540 23050 110 618 645 252 170 183 90 96 91 0 877 920 296 230 243 112 100 98 0 0.692 0.688 0.696 0.64 2.038 0.655 0.558 80 531 1095 1165 -0.53 448 647 475 690 -50 1.1948 0 0.8268016 -0.2743873
6 12960 36530 110 854 1049 634 159 183 192 93 80 0 1840 2183 1096 231 250 111 100 100 0 0.432 0.456 0.446 0.403 1.834 0.484 0.753 80 478 2304 2842 -1.211 695 1609 890 1952 -50 1.1948 0 0.5161536 -0.9541283
7 16730 50040 110 3403 3430 1140 196 226 151 100 100 0 3962 4169 1039 255 299 158 100 100 0 0.865 0.826 0.872 0.77 1.679 0.892 0.845 80 466 6914 7148 -0.209 3207 3707 3234 3914 -50 1.1948 0 1.033502 0.0475413
8 17630 40680 90 1144 1115 343 182 199 119 100 96 0 1503 1494 400 241 256 100 100 100 0 0.762 0.745 0.734 0.723 1.454 0.765 0.772 52 363 2224 2186 -0.392 962 1262 933 1253 -50 1.1948 0 0.9104376 -0.1353683
9 15650 49500 70 876 878 556 169 180 86 87 75 0 1001 1173 774 245 249 60 93 90 0 0.935 0.764 0.671 0.803 2.653 0.696 0.595 32 236 1463 1637 -0.097 707 756 709 928 -50 1.1948 0 1.117138 0.1598073
10 3080 31630 90 2971 3037 836 181 202 132 100 100 0 3620 3347 815 249 290 374 100 100 0 0.828 0.922 0.896 0.914 1.309 0.934 0.897 52 375 6161 5954 -0.273 2790 3371 2856 3098 -50 1.1948 0 0.9892944 -0.0155283
11 13840 27590 100 1069 1076 468 170 179 74 98 97 0 1531 1433 510 238 244 80 100 100 0 0.695 0.758 0.771 0.72 1.511 0.78 0.841 80 450 2192 2101 -0.524 899 1293 906 1195 -50 1.1948 0 0.830386 -0.2681463
12 18140 45210 80 748 836 523 186 198 99 82 75 0 991 1085 548 241 275 467 71 34 0 0.749 0.77 0.736 0.64 2.681 0.377 0.279 52 320 1312 1494 -0.416 562 750 650 844 -50 1.1948 0 0.8949052 -0.1601933
13 10840 23050 100 1282 1250 289 157 164 70 100 100 0 1518 1473 333 213 226 81 100 100 0 0.862 0.867 0.819 0.873 1.493 0.85 0.769 80 440 2430 2353 -0.214 1125 1305 1093 1260 -50 1.1948 0 1.0299176 0.0425293
14 15000 36520 90 514 503 193 161 175 91 86 80 0 863 914 211 231 241 80 100 98 0 0.559 0.501 0.535 0.447 2.101 0.503 0.577 52 361 985 1025 -0.84 353 632 342 683 -50 1.1948 0 0.6678932 -0.5823113
15 16790 27600 80 505 509 183 162 177 78 94 84 0 879 879 221 224 240 85 100 98 0 0.524 0.53 0.534 0.509 1.908 0.571 0.597 52 328 998 1002 -0.933 343 655 347 655 -50 1.1948 0 0.6260752 -0.6755923
16 14200 36540 90 2782 2643 1039 157 206 281 96 88 0 3594 3619 943 225 272 251 100 100 0 0.779 0.732 0.772 0.639 1.879 0.79 0.772 52 355 5994 5880 -0.36 2625 3369 2486 3394 -50 1.1948 0 0.9307492 -0.1035363
17 17520 18610 110 1685 1796 695 181 191 92 100 100 0 5832 6045 2112 238 247 103 100 100 0 0.269 0.278 0.271 0.272 1.261 0.274 0.909 80 492 7098 7422 -1.895 1504 5594 1615 5807 -50 1.1948 0 0.3214012 -1.6375533
18 9700 27200 100 3465 3987 2658 185 195 87 98 95 0 4895 5668 3378 244 257 85 100 100 0 0.705 0.701 0.698 0.61 1.728 0.72 0.916 80 420 7931 9226 -0.504 3280 4651 3802 5424 -50 1.1948 0 0.842334 -0.2475363
19 2360 36350 80 2543 3155 1648 172 203 230 100 100 0 2948 3405 1707 239 253 81 100 100 0 0.875 0.942 0.942 0.929 1.484 0.938 0.777 52 322 5080 6149 -0.192 2371 2709 2983 3166 -50 1.1948 0 1.04545 0.0641243
20 12760 27400 100 4009 3748 1213 165 174 76 100 100 0 4477 4393 1258 227 237 72 100 100 0 0.904 0.86 0.899 0.837 1.421 0.868 0.939 80 442 8094 7749 -0.145 3844 4250 3583 4166 -50 1.1948 0 1.0800992 0.1111643

Total number of rows: 20000

Table truncated, full table size 3826 Kbytes.




Supplementary data files not provided
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