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Sample GSM14987 Query DataSets for GSM14987
Status Public on Jan 31, 2004
Title h1B
Sample type RNA
 
Channel 1
Source name D. simulans Sim-1 adult females
Organism Drosophila simulans
Extracted molecule total RNA
 
Channel 2
Source name adult hybrid females resulting from the cross D. melanogaster Canton S females x D. simulans Sim-1 males
Organism Drosophila melanogaster
Extracted molecule total RNA
 
 
Description Strains of Drosophila melanogaster and Drosophila simulans were raised on glucose-cornmeal-yeast medium at 25 C. Males and virgin females were collected for the interspecific and conspecific crosses. All crosses were performed at 18C using 7-8 individuals from each sex. Virgin females from the parental species as well as hybrid females were stored separately for 5-6 days at room temperature and then snap frozen in liquid nitrogen invariably at the same time of the day in a time window of 2 hours. For each cross, two independent cohorts were used as the source of mRNA. Total RNA was extracted by homogenizing in TRIzol reagent (Invitrogen) followed by a chloroform extraction and isopropanol precipitation. The resulting pellet was washed with 75% ethanol, air-dried, and resuspended in TE. Individual extractions were pooled into one common RNA sample for each strain. Poly-A RNA was purified from total RNA using the Oligotex Direct mRNA kit (Qiagen). mRNA samples were confirmed to be of high quality with a 2100 Bioanalyzer (Agilent). The production of cDNA and hybridizations were done following a published protocol (Eisen and Brown, Methods in Enzymology 303:179-205). 2 µg of poly-A RNA was used as a template for SuperScript II reverse transcriptase (Invitrogen) in the presence of amino-allyl dUTP (Sigma) with random hexamer and oligo dT primers. Reverse transcription was done at 42 C for 3 hours, followed by degradation of RNA by the addition of NaOH and EDTA and incubation at 65 C for 15 minutes. The cDNA was concentrated and purified with Microcon-30 filters (Millipore). Cyanine-3 or cyanine-5 fluorochromes (Amersham) were incorporated with the addition of 1 M NaHCO3 and a 75 minute incubation at room temperature with the cDNA probe. The labeled probes were then purified using the QIAquick PCR Purification Kit (Qiagen), concentrated with Microcon-30 filters, and combined into one sample. 25 µL of probe was combined with 22 µg poly(A), 4.4 µL 20X SSC, 0.71 µL 1 M HEPES (pH 7.0), and 0.66 µL 10% SDS. The probe was boiled at 100 C for 2 minutes and injected under a LifterSlip (Erie Scientific Company) placed over the array. Slides were placed in a Telechem hybridization chamber with 3X SSC in the chamber wells to prevent drying, sealed, and placed in a 65 C water bath for 12 hours. After hybridization, slides were washed with two wash solutions (0.6X SSC, 0.025% SDS [solution 1] and 0.05X SSC [solution 2]). Slides were dried by centrifugation at 1000 rpm for 2 minutes and scanned within an hour of washing. Fluorescence intensity was measured with Axon GenePix 4000A or 4000B scanners and the data was captured with GenePix 4.0 software. Following exclusion of suboptimal spots, intensity ratios were constructed by subtracting local background from foreground for each spot and normalizing one channel so that the mean foreground fluorescence of both channels across the slide was equal.
 
Submission date Jan 07, 2004
Last update date Jun 12, 2013
Contact name Jose M Ranz
E-mail(s) jmr68@mole.bio.cam.ac.uk
Phone 44 1223 766336
URL http://www.gen.cam.ac.uk/
Organization name University of Cambridge
Department Genetics
Lab Michael Ashburner
Street address Downing
City Cambridge
ZIP/Postal code cb2 3eh
Country United Kingdom
 
Platform ID GPL356
Series (1)
GSE948 whole body

Data table header descriptions
ID_REF
X x coordinate position in scanned area of slide (center of spot)
Y y coordinate position in scanned area of slide (center of spot)
DIA diameter of spot
F635_MED Cy5 foreground median pixel intensity
F635_MEAN Cy5 foreground mean pixel intensity
F635_SD Cy5 foreground pixel intensity standard deviation
B635_MED Cy5 background median pixel intensity
B635_MEAN Cy5 background mean pixel intensity
B635_SD Cy5 background pixel intensity standard deviation
%>B635+1SD percent of foreground pixels with a Cy5 intensity greater than (background + 1 standard deviation of background)
%>B635+2SD percent of foreground pixels with a Cy5 intensity greater than (background + 2 standard deviations of background)
F635%SAT percentage of foreground pixels saturated in the Cy5 channel
F532_MED Cy3 foreground median pixel intensity
F532_MEAN Cy3 foreground mean pixel intensity
F532_SD Cy3 foreground pixel intensity standard deviation
B532_MED Cy3 background median pixel intensity
B532_MEAN Cy3 background mean pixel intensity
B532_SD Cy3 background pixel intensity standard deviation
%>B532+1SD percent of foreground pixels with a Cy3 intensity greater than (background + 1 standard deviation of background)
%>B532+2SD percent of foreground pixels with a Cy3 intensity greater than (background + 2 standard deviations of background)
F532%SAT percentage of foreground pixels saturated in the Cy3 channel
RATIO_OF_MEDIANS(635/532) ratio of median pixel intensities, with each pixel intensity corrected by subtracting the median background intensity for that channel
RATIO_OF_MEANS(635/532) ratio of mean pixel intensities, with each pixel intensity corrected by subtracting the median background intensity for that channel
MEDIAN_OF_RATIOS(635/532) median ratio of pixel intensities
MEAN_OF_RATIOS(635/532) mean ratio of pixel intensities
RATIOS_SD(635/532) standard deviation of ratios of pixel intensities
RGN_RATIO(635/532) slope of the least-squares fit linear regression between the two channels, including foreground and background pixels
RGN_R*2(635/532) R*2 for the linear regression
F_PIXELS number of foreground pixels in spot
B_PIXELS number of background pixels in spot
SUM_OF_MEDIANS sum of the background corrected median pixel intensity for Cy5 and Cy3
SUM_OF_MEANS sum of the background corrected mean pixel intensity for Cy5 and Cy3
LOG_RATIO(635/532) log2 of the ratio of medians
F635_MED-B635 Cy5 foreground median intensity minus Cy5 background median intensity
F532_MED-B532 Cy3 foreground median intensity minus Cy3 background median intensity
F635_MEAN-B635 Cy5 foreground mean intensity minus Cy5 background median intensity
F532_MEAN-B532 Cy3 foreground mean intensity minus Cy3 background median intensity
FLAGS quality control identifiers (100 or 0 = good spot, -50 = spot not found by imaging software, -100 = spot flagged as bad by user)
PCR agarose gel quality control of PCR amplification of cDNAs (1 = pass, 0 = fail)
QC_PASS three quality control criteria for inclusion of spots in analysis (1 = pass, 0 = failed), (1) [F635_MEAN-B635] > 2*[B635_SD] or [F532_MEAN-B532] > 2*[B532_SD], (2) spot passed PCR, (3) spot passed FLAGS
POLYMERASE polymerase used to amplify clone (0 - Taq [Amersham], 1 - Herculase [Stratagene])
NORM_F635 [(F635_MEAN-B635)*F_pixels]*SUM[(F532_MEAN-B532)*F_pixels]/SUM[(F635_MEAN-B635)*F_pixels] (SUM denotes the total intensity of fluorescence for a particular channel which is based only on those spots that passed QC_PASS; spots amplified by Taq and Herculase were normalized separately)
VALUE [NORM_F635]/[( F532_MEAN-B532)*F_pixels]

Data table
ID_REF X Y DIA F635_MED F635_MEAN F635_SD B635_MED B635_MEAN B635_SD %>B635+1SD %>B635+2SD F635%SAT F532_MED F532_MEAN F532_SD B532_MED B532_MEAN B532_SD %>B532+1SD %>B532+2SD F532%SAT RATIO_OF_MEDIANS(635/532) RATIO_OF_MEANS(635/532) MEDIAN_OF_RATIOS(635/532) MEAN_OF_RATIOS(635/532) RATIOS_SD(635/532) RGN_RATIO(635/532) RGN_R*2(635/532) F_PIXELS B_PIXELS SUM_OF_MEDIANS SUM_OF_MEANS LOG_RATIO(635/532) F635_MED-B635 F532_MED-B532 F635_MEAN-B635 F532_MEAN-B532 FLAGS PCR QC_PASS POLYMERASE NORM_F635 VALUE
1.1.1 1340 39410 70 871 996 641 125 127 26 100 100 0 974 1154 768 164 169 33 98 98 0 0.921 0.88 0.844 0.919 1.852 0.813 0.753 156 1140 1556 1861 -0.119 746 810 871 990 0 1 1 0 125419.6702 0.812093176
1.1.2 1535 39405 70 876 1041 563 124 126 26 100 100 0 598 669 335 172 174 32 97 92 0 1.765 1.845 1.848 2.031 1.812 1.851 0.847 156 1130 1178 1414 0.82 752 426 917 497 0 1 1 0 132043.4415 1.703083133
1.1.3 1740 39405 75 1420 1847 1209 132 133 28 100 100 0 724 859 450 171 172 33 98 98 0 2.329 2.493 2.393 2.505 1.69 2.735 0.821 156 1272 1841 2403 1.22 1288 553 1715 688 0 1 1 0 246951.4745 2.300904466
1.1.4 1940 39400 70 1298 1426 607 144 147 37 100 100 0 964 1026 348 183 185 36 100 100 0 1.478 1.521 1.492 1.505 1.468 1.638 0.849 156 1114 1935 2125 0.563 1154 781 1282 843 0 1 1 0 184601.627 1.403729256
1.1.5 2135 39395 70 5105 5616 2514 148 149 30 100 100 0 6721 7127 2967 174 177 34 100 100 0 0.757 0.786 0.778 0.781 1.347 0.791 0.876 156 1094 11504 12421 -0.401 4957 6547 5468 6953 0 1 1 0 787364.8179 0.72590398
1.1.6 2335 39395 70 1211 1394 615 146 148 28 100 100 0 1318 1482 594 188 188 33 100 100 0 0.942 0.964 0.933 0.96 1.365 0.977 0.916 156 1094 2195 2542 -0.085 1065 1130 1248 1294 0 1 1 0 179705.796 0.890232018
1.1.7 2535 39395 65 16809 17300 5577 147 162 92 100 100 0 12808 13759 4300 195 204 77 100 100 0 1.321 1.265 1.252 1.26 1.221 1.297 0.915 120 914 29275 30717 0.402 16662 12613 17153 13564 0 1 1 0 1899959.023 1.167280438
1.1.8 2745 39395 70 2134 2482 1563 149 151 28 100 100 0 1553 1754 954 187 191 56 100 100 0 1.453 1.489 1.483 1.454 1.59 1.584 0.882 156 1094 3351 3900 0.539 1985 1366 2333 1567 0 1 1 0 335940.4024 1.374259169
1.1.9 2935 39395 75 973 1401 972 152 154 28 100 100 0 770 921 509 178 181 36 100 100 0 1.387 1.681 1.644 1.628 1.632 1.862 0.806 156 1246 1413 1992 0.472 821 592 1249 743 0 1 1 0 179849.7911 1.55165986
1.1.10 3130 39395 70 1681 2039 1328 152 154 27 100 100 0 575 640 316 191 191 34 99 98 0 3.982 4.203 4.03 4.255 1.712 4.746 0.771 156 1094 1913 2336 1.993 1529 384 1887 449 0 0 0 0
1.1.11 3335 39395 70 5136 5687 2669 155 157 27 100 100 0 3858 4288 1927 191 193 35 100 100 0 1.358 1.35 1.377 1.342 1.402 1.39 0.9 156 1094 8648 9629 0.442 4981 3667 5532 4097 0 1 1 0 796580.4998 1.24634739
1.1.12 3535 39395 70 5841 6547 3539 156 158 27 100 100 0 6249 6650 3466 193 196 34 100 100 0 0.939 0.99 1.012 0.991 1.474 1.001 0.889 156 1094 11741 12848 -0.091 5685 6056 6391 6457 0 1 1 0 920272.2296 0.913610184
1.1.13 3735 39390 75 11596 13182 6462 163 166 34 100 100 0 10628 11456 5941 190 195 47 100 100 0 1.095 1.156 1.135 1.198 1.582 1.112 0.885 156 1226 21871 24285 0.131 11433 10438 13019 11266 0 0 0 0
1.1.14 3935 39395 75 5166 5869 3265 166 169 39 100 100 0 4729 5404 3089 191 194 45 100 100 0 1.102 1.094 1.068 1.114 1.458 1.074 0.886 156 1246 9538 10916 0.14 5000 4538 5703 5213 0 1 1 0 821203.6497 1.009807397
1.1.15 4135 39400 75 1291 1438 717 162 167 114 100 100 0 3796 4277 2216 198 203 61 100 100 0 0.314 0.313 0.297 0.315 1.602 0.291 0.795 156 1262 4727 5355 -1.672 1129 3598 1276 4079 0 1 1 0 183737.6568 0.288748589
1.1.16 4335 39395 75 8373 9446 4911 168 170 41 100 100 0 364 380 111 203 205 34 95 83 0 50.963 52.418 49.89 47.464 1.519 55.69 0.716 156 1246 8366 9455 5.671 8205 161 9278 177 0 1 1 0 1335985.878 48.3842488
1.1.17 4545 39400 70 3258 3550 1533 163 163 30 100 100 0 1075 1122 425 202 205 36 100 100 0 3.545 3.682 3.696 3.693 1.478 3.875 0.882 156 1114 3968 4307 1.826 3095 873 3387 920 0 1 1 0 487711.1628 3.398210443
1.1.18 4740 39400 70 2358 2581 983 166 168 29 100 100 0 2220 2341 877 208 210 37 100 100 0 1.089 1.132 1.114 1.144 1.384 1.136 0.904 156 1114 4204 4548 0.124 2192 2012 2415 2133 0 1 1 0 347747.9947 1.045079143
1.1.19 4935 39405 65 2940 3451 1972 173 175 36 100 100 0 2028 2261 1127 208 213 42 100 100 0 1.52 1.597 1.569 1.565 1.404 1.698 0.869 120 940 4587 5331 0.604 2767 1820 3278 2053 0 1 1 0 363089.0036 1.473814757
1.1.20 5135 39400 70 4334 4956 2758 170 172 30 100 100 0 9222 10496 6355 209 213 41 100 100 0 0.462 0.465 0.456 0.495 1.628 0.434 0.872 156 1114 13177 15073 -1.114 4164 9013 4786 10287 0 1 1 0 689160.2082 0.429444312

Total number of rows: 6101

Table truncated, full table size 1169 Kbytes.




Supplementary data files not provided
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