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Sample GSM4617 Query DataSets for GSM4617
Status Public on Jul 25, 2003
Title MGRef Cy3 vs 12APFc Cy5 630600GEO
Sample type RNA
 
Channel 1
Source name 635nm (Cy5) channel, midgut at time point 12 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 2010 15940 100 95 225 335 72 74 25 42 32 0 110 241 324 90 93 20 48 30 0 1.15 1.013 1.261 1.189 2.563 1.043 0.658 80 480 43 304 0.202 23 20 153 151 -75 1.18544 0 1.363256 0.4470573
2 2190 15940 100 88 126 116 72 74 23 43 27 0 99 152 151 92 92 18 37 28 0 2.286 0.9 1.503 1.264 4.48 0.736 0.483 80 444 23 114 1.193 16 7 54 60 -75 1.18544 0 2.70991584 1.4382483
3 2360 15940 100 89 127 156 71 73 24 45 21 0 102 163 257 94 94 19 38 26 0 2.25 0.812 0.84 1.269 3.751 0.595 0.739 80 432 26 125 1.17 18 8 56 69 -75 1.18544 0 2.66724 1.4153483
4 2530 15940 100 89 160 245 74 77 29 36 17 0 99 210 385 99 100 23 21 13 0 100000 0.775 1 1.088 3.57 0.619 0.786 80 420 15 197 16.61 15 0 86 111 -75 1.18544 0 118544 16.8550633
5 2700 15940 100 77 104 113 77 80 30 16 8 0 108 144 187 99 102 24 27 15 0 0 0.6 0.762 0.736 5.297 0.611 0.621 80 442 9 72 Error 0 9 27 45 -75 1.18544 0 0
6 2870 15940 100 93 205 322 75 78 30 35 28 0 109 245 414 101 102 21 38 27 0 2.25 0.903 0.923 1.152 3.572 0.763 0.757 80 432 26 274 1.17 18 8 130 144 -75 1.18544 0 2.66724 1.4153483
7 3040 15940 100 91 371 848 77 81 31 28 21 0 115 350 795 101 104 26 26 18 0 1 1.181 0.988 1.179 3.181 1.083 0.825 80 432 28 543 0 14 14 294 249 -75 1.18544 0 1.18544 0.2454233
8 3210 15940 100 91 103 87 79 82 28 25 12 0 101 118 87 98 100 26 18 6 0 4 1.2 0.957 1.022 3.629 0.846 0.629 80 420 15 44 2 12 3 24 20 -75 1.18544 0 4.74176 2.2454233
9 3380 15940 100 91 333 659 71 74 27 43 28 0 90 394 846 88 89 19 33 26 0 10 0.856 1.546 2.024 4.976 0.775 0.853 80 432 22 568 3.322 20 2 262 306 -75 1.18544 0 11.8544 3.5673513
10 3550 15940 100 88 197 326 67 70 26 46 26 0 94 231 430 88 89 18 38 22 0 3.5 0.909 1.125 1.382 3.97 0.751 0.793 80 442 27 273 1.807 21 6 130 143 -75 1.18544 0 4.14904 2.0527783
11 3720 15940 100 72 170 333 71 75 27 33 20 0 96 236 526 88 89 18 35 25 0 0.125 0.669 1.4 1.477 3.693 0.609 0.802 80 432 9 247 -3 1 8 99 148 -75 1.18544 0 0.14818 -2.7545773
12 3890 15940 100 83 186 351 71 73 24 38 26 0 96 250 535 87 89 19 32 23 0 1.333 0.706 1.453 1.326 2.932 0.631 0.787 80 432 21 278 0.415 12 9 115 163 -75 1.18544 0 1.58019152 0.6600993
13 4060 15940 100 73 154 256 70 73 25 25 18 0 84 190 341 87 88 18 23 18 0 -1 0.816 0.896 0.912 2.599 0.736 0.822 80 426 0 187 Error 3 -3 84 103 -75 1.18544 0 -1.18544
14 4230 15940 100 78 117 129 68 73 25 40 27 0 91 144 218 86 88 21 26 13 0 2 0.845 0.938 0.817 3.732 0.565 0.662 80 428 15 107 1 10 5 49 58 -75 1.18544 0 2.37088 1.2454233
15 4400 15950 100 90 194 304 66 69 23 51 32 0 94 273 505 86 88 21 36 23 0 3 0.684 1.034 1.229 4.008 0.582 0.821 80 442 32 315 1.585 24 8 128 187 -75 1.18544 0 3.55632 1.8303853
16 4570 15950 100 72 202 348 66 68 23 41 26 0 86 250 452 83 85 17 31 25 0 2 0.814 0.882 1.141 4.014 0.764 0.905 80 420 9 303 1 6 3 136 167 -75 1.18544 0 2.37088 1.2454233
17 4740 15950 100 86 134 155 66 67 23 42 33 0 96 160 197 83 85 18 37 23 0 1.538 0.883 1.295 1.337 2.803 0.787 0.801 80 432 33 145 0.621 20 13 68 77 -75 1.18544 0 1.82320672 0.8664783
18 4910 15950 100 75 122 156 67 71 30 27 16 0 83 148 222 83 84 17 23 18 0 100000 0.846 1.1 1.154 3.684 0.7 0.786 80 460 8 120 16.61 8 0 55 65 -75 1.18544 0 118544 16.8550633
19 5080 15950 100 72 75 28 68 72 25 22 7 0 85 88 24 82 84 18 25 11 0 1.333 1.167 1.356 0.966 4.773 1.557 0.05 80 442 7 13 0.415 4 3 7 6 -75 1.18544 0 1.58019152 0.6600993
20 5250 15950 100 74 153 222 67 72 29 28 20 0 84 180 275 81 82 18 33 20 0 2.333 0.869 1.29 1.399 4.161 0.814 0.852 80 420 10 185 1.222 7 3 86 99 -75 1.18544 0 2.76563152 1.4676093

Total number of rows: 20000

Table truncated, full table size 3812 Kbytes.




Supplementary data files not provided
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