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Sample GSM4605 Query DataSets for GSM4605
Status Public on Jul 25, 2003
Title MGRef Cy3 vs 5APFc Cy5 630650GEO
Sample type RNA
 
Channel 1
Source name 635nm (Cy5) channel, midgut at time point 5 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_Dmel20010730arrayplatform
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 2070 15770 100 145 309 403 120 127 50 33 27 0 134 284 423 121 125 42 25 22 0 1.923 1.16 1.021 1.056 3.028 0.984 0.802 80 468 38 352 0.943 25 13 189 163 -75 1.08744 0 2.09114712 1.0642953
2 2240 15770 100 155 208 170 123 130 37 45 31 0 139 192 153 120 123 34 35 25 0 1.684 1.181 1.368 1.425 3.915 1.074 0.415 80 432 51 157 0.752 32 19 85 72 -75 1.08744 0 1.83124896 0.8728283
3 2410 15770 100 144 360 453 122 129 61 43 30 0 158 333 425 120 124 32 52 35 0 0.579 1.117 1.12 0.979 2.551 1.083 0.86 80 432 60 451 -0.788 22 38 238 213 -75 1.08744 0 0.62962776 -0.6674293
4 2580 15770 100 139 253 291 116 121 43 43 32 0 139 236 265 117 123 63 26 20 0 1.045 1.151 0.998 0.952 2.801 1.118 0.758 80 432 45 256 0.064 23 22 137 119 -75 1.08744 0 1.1363748 0.1844393
5 2750 15770 100 167 379 436 120 125 40 52 38 0 152 349 433 121 126 63 28 23 0 1.516 1.136 1.545 1.616 2.788 1.002 0.808 80 432 78 487 0.6 47 31 259 228 -75 1.08744 0 1.64855904 0.7212063
6 2920 15770 100 151 487 715 119 123 37 47 38 0 134 442 655 122 126 35 38 31 0 2.667 1.15 1.311 1.282 4.379 1.113 0.799 80 432 44 688 1.415 32 12 368 320 -75 1.08744 0 2.90020248 1.5361543
7 3090 15780 100 128 428 594 117 121 35 40 32 0 140 367 498 122 125 33 37 31 0 0.611 1.269 1.1 0.99 3.622 1.202 0.818 80 432 29 556 -0.71 11 18 311 245 -75 1.08744 0 0.66442584 -0.5898203
8 3260 15780 100 183 632 956 130 134 50 52 38 0 152 508 774 124 127 34 45 40 0 1.893 1.307 1.361 1.645 3.135 1.289 0.727 80 432 81 886 0.921 53 28 502 384 -75 1.08744 0 2.05852392 1.0416103
9 3430 15780 100 172 487 690 138 141 36 50 33 0 156 464 643 129 130 33 43 33 0 1.259 1.042 0.989 0.95 3.319 1.085 0.86 80 442 61 684 0.333 34 27 349 335 -75 1.08744 0 1.36908696 0.4532143
10 3600 15780 100 133 386 514 123 126 37 37 33 0 129 328 485 122 125 30 32 26 0 1.429 1.277 1.429 1.631 3.452 1.026 0.706 80 432 17 469 0.515 10 7 263 206 -75 1.08744 0 1.55395176 0.6359423
11 3770 15780 100 118 321 474 114 128 125 23 21 0 134 314 431 118 130 116 23 21 0 0.25 1.056 1.08 1.074 4.473 1.082 0.75 80 432 20 403 -2 4 16 207 196 -75 1.08744 0 0.27186 -1.8790643
12 3940 15790 100 122 386 589 103 112 58 31 28 0 128 343 498 114 122 54 32 26 0 1.357 1.236 1.157 1.04 3 1.141 0.813 80 428 33 512 0.441 19 14 283 229 -75 1.08744 0 1.47565608 0.5613573
13 4110 15790 100 125 427 642 106 109 32 43 30 0 130 406 621 117 121 30 36 28 0 1.462 1.111 1.056 1.504 4.298 1.018 0.681 80 437 32 610 0.547 19 13 321 289 -75 1.08744 0 1.58983728 0.6688793
14 4280 15790 100 123 388 592 104 107 30 42 31 0 126 346 490 115 118 29 33 30 0 1.727 1.229 1.361 1.383 3.898 1.182 0.722 80 451 30 515 0.788 19 11 284 231 -75 1.08744 0 1.87800888 0.9092043
15 4450 15790 100 119 543 865 104 112 76 32 27 0 131 501 737 117 122 38 33 31 0 1.071 1.143 1.142 1.057 2.981 1.163 0.83 80 451 29 823 0.1 15 14 439 384 -75 1.08744 0 1.16464824 0.2198943
16 4620 15790 100 109 474 873 101 111 77 27 23 0 125 403 666 118 122 36 33 25 0 1.143 1.309 1.284 1.164 3.875 1.248 0.817 80 432 15 658 0.193 8 7 373 285 -75 1.08744 0 1.24294392 0.3137613
17 4790 15790 100 121 248 361 106 163 266 12 8 0 119 226 293 115 159 217 15 10 0 3.75 1.279 0.917 0.996 3.656 1.271 0.765 80 442 19 253 1.907 15 4 142 111 -75 1.08744 0 4.0779 2.0278263
18 4960 15800 100 106 115 40 106 161 264 0 0 0 112 114 33 113 160 215 0 0 0 0 9 1.24 1.107 5.2 1.298 0.841 80 450 -1 10 Error 0 -1 9 1 -75 1.08744 0 0
19 5130 15800 100 128 326 435 102 108 56 28 26 0 116 282 371 108 116 65 27 22 0 3.25 1.287 1.216 1.482 2.475 1.163 0.758 80 440 34 398 1.7 26 8 224 174 -75 1.08744 0 3.53418 1.8213763
20 5300 15800 100 110 328 540 103 117 82 30 23 0 115 275 422 111 123 86 21 17 0 1.75 1.372 1.569 1.406 2.697 1.231 0.8 80 432 11 389 0.807 7 4 225 164 -75 1.08744 0 1.90302 0.9282913

Total number of rows: 20000

Table truncated, full table size 3798 Kbytes.




Supplementary data files not provided
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