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Sample GSM4599 Query DataSets for GSM4599
Status Public on Jul 25, 2003
Title MGRef Cy3 vs 3APFc Cy5 630630GEO
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_Dmel20010730arrayplatform
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 1940 15930 100 97 207 335 99 110 73 16 13 0 135 234 323 119 132 99 15 12 0 -0.125 0.939 1.217 1.17 3.74 0.938 0.679 80 464 14 223 Error -2 16 108 115 -75 1.03628 0 -0.129535
2 2110 15930 100 102 118 69 97 107 76 7 3 0 128 139 48 122 135 104 2 2 0 0.833 1.235 1.1 1.008 3.856 1.018 0.535 80 420 11 38 -0.263 5 6 21 17 -75 1.03628 0 0.86322124 -0.2121983
3 2280 15930 100 129 151 99 100 105 56 23 16 0 132 163 117 121 130 78 13 6 0 2.636 1.214 1.343 1.236 4.083 0.874 0.296 80 432 40 93 1.399 29 11 51 42 -75 1.03628 0 2.73163408 1.4497643
4 2450 15930 100 107 122 81 101 110 71 6 6 0 124 135 63 120 130 82 3 3 0 1.5 1.4 1 0.994 4.083 0.995 0.531 80 432 10 36 0.585 6 4 21 15 -75 1.03628 0 1.55442 0.6363763
5 2620 15930 100 97 128 146 97 104 54 11 6 0 119 157 134 119 121 35 20 11 0 0 0.816 1.184 1.132 3.034 1.017 0.678 80 442 0 69 Error 0 0 31 38 -75 1.03628 0 0
6 2790 15930 100 106 130 123 100 123 135 3 2 0 133 189 239 124 140 128 7 6 0 0.667 0.462 0.788 0.766 4.074 0.966 0.697 80 420 15 95 -0.585 6 9 30 65 -75 1.03628 0 0.69119876 -0.5328273
7 2960 15930 100 118 273 416 103 125 134 18 13 0 129 264 411 123 140 128 12 12 0 2.5 1.206 1.324 1.69 5.952 0.966 0.597 80 420 21 311 1.322 15 6 170 141 -75 1.03628 0 2.5907 1.3733423
8 3130 15930 100 113 382 568 102 105 31 42 32 0 143 426 601 124 128 28 47 32 0 0.579 0.927 1.053 1.192 3.378 0.96 0.683 80 420 30 582 -0.788 11 19 280 302 -75 1.03628 0 0.60000612 -0.7369513
9 3300 15930 100 127 282 327 103 108 37 41 32 0 151 312 342 126 129 30 43 36 0 0.96 0.962 0.993 0.861 2.658 0.962 0.651 80 432 49 365 -0.059 24 25 179 186 -75 1.03628 0 0.9948288 -0.0074803
10 3470 15930 100 123 185 219 101 106 38 31 12 0 138 199 223 125 129 29 35 15 0 1.692 1.135 1.176 1.119 3.61 0.889 0.668 80 432 35 158 0.759 22 13 84 74 -75 1.03628 0 1.75338576 0.8101433
11 3640 15930 100 107 115 70 102 111 55 8 1 0 138 148 80 127 135 45 12 6 0 0.455 0.619 1.059 0.947 5.888 1.232 0.622 80 432 16 34 -1.138 5 11 13 21 -75 1.03628 0 0.4715074 -1.0846483
12 3810 15930 100 99 121 85 109 130 97 6 3 0 127 157 147 129 148 102 6 3 0 5 0.429 1.245 1.69 3.358 0.905 0.685 80 420 -12 40 2.322 -10 -2 12 28 -75 1.03628 0 5.1814 2.3733423
13 3980 15930 100 109 236 367 106 124 92 18 13 0 127 236 332 124 139 94 12 11 0 1 1.161 1.105 1.143 4.305 1.039 0.74 80 442 6 242 0 3 3 130 112 -75 1.03628 0 1.03628 0.0514143
14 4150 15930 100 121 148 122 105 113 60 18 10 0 143 173 127 128 133 41 26 13 0 1.067 0.956 1.364 1.218 3.304 0.811 0.501 80 432 31 88 0.093 16 15 43 45 -75 1.03628 0 1.10571076 0.1449743
15 4320 15930 100 117 171 201 104 113 61 22 7 0 136 204 231 132 136 40 26 12 0 3.25 0.931 1.096 0.901 3.115 0.989 0.689 80 442 17 139 1.7 13 4 67 72 -75 1.03628 0 3.36791 1.7518543
16 4490 15930 100 146 512 950 104 107 30 56 42 0 147 534 1022 131 133 27 42 31 0 2.625 1.012 1.213 1.183 4.418 0.926 0.66 80 444 58 811 1.392 42 16 408 403 -75 1.03628 0 2.720235 1.4437313
17 4670 15930 100 115 129 61 109 157 221 1 0 0 140 147 62 134 171 198 1 1 0 1 1.538 1.01 0.94 3.528 0.923 0.727 80 444 12 33 0 6 6 20 13 -75 1.03628 0 1.03628 0.0514143
18 4840 15930 100 98 102 28 112 164 233 0 0 0 127 130 29 135 171 197 0 0 0 1.75 2 0.931 1.075 2.855 1.151 0.539 80 450 -22 -15 0.807 -14 -8 -10 -5 -75 1.03628 0 1.81349 0.8587693
19 5010 15930 100 112 160 220 105 131 150 6 5 0 128 211 333 130 146 123 7 6 0 -3.5 0.679 0.938 1.051 3.965 0.933 0.688 80 432 5 136 Error 7 -2 55 81 -75 1.03628 0 -3.62698
20 5180 15930 100 115 182 311 107 148 208 7 5 0 125 194 315 130 160 174 5 3 0 -1.6 1.172 0.858 1.03 4.071 0.968 0.682 80 432 3 139 Error 8 -5 75 64 -75 1.03628 0 -1.658048

Total number of rows: 20000

Table truncated, full table size 3911 Kbytes.




Supplementary data files not provided
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