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Sample GSM7878 Query DataSets for GSM7878
Status Public on Jul 16, 2003
Title E85a
Sample type RNA
 
Channel 1
Source name Zimbabwe53 adult males
Organism Drosophila melanogaster
Extracted molecule total RNA
 
Channel 2
Source name St Louis adult males
Organism Drosophila melanogaster
Extracted molecule total RNA
 
 
Description Strains of Drosophila melanogaster were raised on standard medium at 25 C with no light/dark cycle. Adult males were collected up to 24 hours after eclosing and separated from females where they were allowed to age an additional 3-4 days. Total RNA was extracted by homogenizing 8-12 flies 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 Jul 14, 2003
Last update date Mar 20, 2006
Contact name Colin Meiklejohn
E-mail(s) [email protected]
Phone 617-496-5540
Fax 617-496-5854
URL http://www.oeb.harvard.edu/hartl/lab/
Organization name Harvard University
Department Organismic and Evolutionary Biology
Lab Hartl Lab
Street address 16 Divinity Ave
City Cambridge
State/province MA
ZIP/Postal code 02138
Country USA
 
Platform ID GPL356
Series (1)
GSE539 Intraspecific variation in gene expression

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 six quality control criteria for inclusion of spots in analysis (1 = pass, 0 = failed), (1) [F635_MEAN-B635] > 3*[B635_SD] or [F532_MEAN-B532] > 3*[B532_SD], (2) [%>B635+2SD] > 70 and [%>B532+2SD] > 70, (3) [F635%SAT] < 10 and [F532%SAT] < 10, (4) [B635_MED] < 3*[B532_MED] and [B532_MED] < 3*[B635_MED], (5) spot passed PCR, (6) spot passed FLAGS
POLYMERASE polymerase used to amplify clone (0 - Taq [Amersham], 1 - Herculase [Stratagene])
NORM_F635 [F635_MEAN-B635]*mean[F532_MEAN-B532]/mean[F635_MEAN-B635] (means were calculated only including spots that passed QC_PASS, spots amplified by Taq and Herculase were normalized separately)
VALUE log ratio of PRE_VALUE
PRE_VALUE [NORM_F635]/[ F635_MEAN-B635]

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 PRE_VALUE
1.1.1 2500 12605 65 5950 5269 1974 125 130 42 100 100 0 5205 4743 1373 126 131 36 100 100 0 1.147 1.114 1.137 1.055 1.417 1.158 0.934 120 930 10904 9761 0.198 5825 5079 5144 4617 0 1 1 0 5652.942868 0.0879153 1.224375756
1.1.10 4300 12600 65 5213 5011 1097 137 140 37 100 100 0 4595 4645 976 138 145 77 100 100 0 1.139 1.081 1.081 1.075 1.157 1.1 0.961 120 930 9533 9381 0.188 5076 4457 4874 4507 0 0 0 0
1.1.11 4500 12610 70 15067 14269 3800 135 140 35 100 100 0 19592 18160 5639 140 144 31 100 100 0 0.768 0.784 0.776 0.812 1.307 0.767 0.963 156 1134 34384 32154 -0.382 14932 19452 14134 18020 0 1 1 0 15532.40562 -0.0645163 0.861953697
1.1.12 4700 12605 80 20005 18636 12493 137 140 34 100 100 0 21277 17214 11399 142 144 30 100 100 0 0.94 1.084 1.116 1.156 1.453 1.09 0.949 208 1484 41003 35571 -0.089 19868 21135 18499 17072 0 1 1 0 20329.2749 0.0758373 1.190796328
1.1.13 4900 12605 70 20691 18851 8532 140 143 37 100 100 0 18000 16065 6955 143 147 33 100 100 0 1.151 1.175 1.165 1.177 1.326 1.191 0.953 156 1114 38408 34633 0.203 20551 17857 18711 15922 0 0 0 0
1.1.14 5100 12605 70 9771 8763 3457 140 144 36 100 100 0 14040 12131 4852 144 147 32 100 100 0 0.693 0.719 0.741 0.744 1.299 0.713 0.969 156 1114 23527 20610 -0.529 9631 13896 8623 11987 0 1 1 0 9476.152089 -0.1020783 0.790535755
1.1.15 5300 12610 75 15158 14588 6231 139 145 49 100 100 0 13287 13077 4262 146 150 38 100 100 0 1.143 1.117 1.13 1.056 1.432 1.172 0.942 156 1272 28160 27380 0.193 15019 13141 14449 12931 0 1 1 0 15878.57144 0.0891793 1.227946133
1.1.16 5490 12615 75 3651 3440 1306 136 141 36 100 100 0 5117 4735 1708 145 149 36 100 100 0 0.707 0.72 0.743 0.732 1.555 0.717 0.939 156 1272 8487 7894 -0.5 3515 4972 3304 4590 0 1 1 0 3630.894875 -0.1017993 0.791044635
1.1.17 5695 12615 65 2497 2536 878 137 142 38 100 100 0 2373 2190 675 146 151 39 100 100 0 1.06 1.174 1.197 1.184 1.437 1.194 0.922 120 940 4587 4443 0.084 2360 2227 2399 2044 0 1 1 0 2636.354965 0.1105233 1.289801842
1.1.18 5895 12605 65 6187 5977 1216 139 147 54 100 100 0 5010 4855 776 147 155 49 100 100 0 1.244 1.24 1.25 1.226 1.133 1.263 0.968 120 930 10911 10546 0.315 6048 4863 5838 4708 0 1 1 0 6415.606622 0.1344013 1.362703191
1.1.19 6095 12610 80 4059 4314 3451 138 139 32 100 98 0 5208 4659 3723 146 149 31 100 99 0 0.775 0.925 0.959 0.964 1.519 0.924 0.938 208 1502 8983 8689 -0.368 3921 5062 4176 4513 0 1 1 0 4589.169793 0.0072693 1.016877862
1.1.2 2705 12610 65 2295 2124 698 128 132 35 100 100 0 1989 1849 554 128 131 31 100 100 0 1.164 1.16 1.176 1.161 1.468 1.176 0.924 120 940 4028 3717 0.22 2167 1861 1996 1721 0 1 1 0 2193.482497 0.1053533 1.27453951
1.1.20 6295 12605 80 13941 13884 11704 136 137 33 100 100 0 12500 13033 11030 147 150 31 99 99 0 1.118 1.067 1.04 1.07 1.415 1.065 0.96 208 1484 26158 26634 0.16 13805 12353 13748 12886 0 1 1 0 15108.21511 0.0690953 1.172451895
1.1.21 6490 12610 65 2556 2316 651 136 141 38 100 100 0 2709 2601 479 151 154 35 100 100 0 0.946 0.89 0.932 0.858 1.366 0.918 0.914 120 940 4978 4630 -0.08 2420 2558 2180 2450 0 1 1 0 2395.687296 -0.0097363 0.977831549
1.1.3 2905 12605 65 5285 4886 1592 126 186 645 98 92 0 4890 4419 1496 131 181 501 98 92 0 1.084 1.11 1.085 1.134 1.327 1.111 0.964 120 940 9918 9048 0.116 5159 4759 4760 4288 0 1 1 0 5230.950243 0.0863263 1.219904441
1.1.4 3105 12605 60 5086 4968 825 132 141 52 100 100 0 5614 5346 1035 132 140 51 100 100 0 0.904 0.928 0.921 0.939 1.177 0.912 0.974 120 810 10436 10050 -0.146 4954 5482 4836 5214 0 1 1 0 5314.469617 0.0082893 1.019269202
1.1.5 3315 12605 80 13753 12133 8042 132 136 44 100 100 0 10458 8307 5523 132 135 34 100 100 0 1.319 1.468 1.458 1.541 1.426 1.475 0.956 208 1502 23947 20176 0.4 13621 10326 12001 8175 0 1 1 0 13188.36846 0.2077033 1.613256081
1.1.6 3505 12595 70 3949 3551 1264 133 136 33 100 100 0 3737 3296 1196 133 136 27 100 100 0 1.059 1.081 1.076 1.099 1.228 1.076 0.978 156 1094 7420 6581 0.082 3816 3604 3418 3163 0 1 1 0 3756.173935 0.0746473 1.187535231
1.1.7 3710 12600 75 19651 17313 8527 131 136 45 100 100 0 16773 14227 7016 133 138 37 100 100 0 1.173 1.219 1.213 1.225 1.239 1.226 0.974 156 1262 36160 31276 0.23 19520 16640 17182 14094 0 1 1 0 18881.97207 0.1270133 1.339717048
1.1.8 3905 12605 75 7960 7764 4637 133 136 33 100 100 0 8411 7203 4142 134 138 32 100 100 0 0.946 1.08 1.084 1.094 1.32 1.093 0.966 156 1272 16104 14700 -0.081 7827 8277 7631 7069 0 1 1 0 8386.004476 0.0741973 1.186307041

Total number of rows: 6101

Table truncated, full table size 1212 Kbytes.




Supplementary data files not provided
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