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Sample GSM7864 Query DataSets for GSM7864
Status Public on Jul 16, 2003
Title C123b
Sample type RNA
 
Channel 1
Source name Canton S adult males
Organism Drosophila melanogaster
Extracted molecule total RNA
 
Channel 2
Source name Zimbabwe53 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 � Cy5 background median intensity
F532_MED-B532 Cy3 foreground median intensity � Cy3 background median intensity
F635_MEAN-B635 Cy5 foreground mean intensity � Cy5 background median intensity
F532_MEAN-B532 Cy3 foreground mean intensity � 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 1240 40450 80 2972 2653 1822 103 109 35 100 98 0 2119 2026 1086 105 108 18 100 100 0 1.425 1.327 1.185 1.14 1.727 1.487 0.855 52 332 4883 4471 0.51 2869 2014 2550 1921 0 1 1 0 1883.08456 -0.0086583 0.980262655
1.1.10 3040 40450 80 2033 2256 1464 116 123 44 100 100 0 1550 1491 777 116 119 25 100 100 0 1.337 1.556 1.495 1.447 1.641 1.749 0.82 52 332 3351 3515 0.419 1917 1434 2140 1375 0 0 0 0
1.1.11 3240 40440 80 17879 15926 7465 118 122 33 100 100 0 12564 11340 4892 118 121 32 100 100 0 1.427 1.409 1.41 1.351 1.508 1.452 0.949 52 320 30207 27030 0.513 17761 12446 15808 11222 0 1 1 0 11673.64735 0.0171363 1.0402466
1.1.12 3440 40440 80 26363 23007 10556 114 122 39 100 100 0 20312 17718 7225 119 120 18 100 100 0 1.3 1.301 1.269 1.211 1.575 1.35 0.935 52 320 46442 40492 0.378 26249 20193 22893 17599 0 1 1 0 16905.66857 -0.0174563 0.96060393
1.1.13 3640 40440 80 15459 14118 6663 110 113 27 100 100 0 16211 14137 5606 114 115 14 100 100 0 0.954 0.999 0.957 0.887 1.705 1.041 0.915 52 320 31446 28031 -0.069 15349 16097 14008 14023 0 0 0 0
1.1.14 3840 40440 90 11202 11182 6031 125 185 303 98 98 0 8176 7824 3043 120 142 81 100 100 0 1.375 1.435 1.361 1.242 1.75 1.58 0.893 52 412 19133 18761 0.459 11077 8056 11057 7704 0 1 1 0 8165.202347 0.0252513 1.05986531
1.1.15 4040 40450 90 10662 10826 6685 122 126 30 100 100 0 7595 7326 3325 115 117 16 100 100 0 1.409 1.484 1.394 1.261 1.675 1.642 0.928 52 420 18020 17915 0.495 10540 7480 10704 7211 0 1 1 0 7904.524367 0.0398803 1.096175893
1.1.16 4250 40440 80 3919 4197 1777 134 139 43 100 100 0 2705 2449 956 115 117 24 100 100 0 1.461 1.741 1.7 1.765 1.398 1.81 0.923 52 320 6375 6397 0.547 3785 2590 4063 2334 0 1 1 0 3000.3814 0.1090763 1.285510454
1.1.17 4440 40440 80 2545 2841 1749 169 233 825 88 73 0 2385 2402 871 117 134 172 100 100 0 1.048 1.169 1.034 0.918 2.008 1.39 0.845 52 320 4644 4957 0.067 2376 2268 2672 2285 0 1 1 0 1973.177234 -0.0637203 0.863534894
1.1.18 4640 40440 80 4045 3817 2000 152 161 91 92 92 0 3199 3152 1215 120 126 55 100 100 0 1.264 1.209 1.205 0.957 2.052 1.36 0.894 52 320 6972 6697 0.338 3893 3079 3665 3032 0 1 1 0 2706.472515 -0.0493263 0.892636054
1.1.19 4840 40450 80 5904 5495 2597 132 138 49 100 100 0 4460 4340 1521 118 119 17 100 100 0 1.329 1.27 1.254 1.159 1.533 1.367 0.936 52 332 10114 9585 0.411 5772 4342 5363 4222 0 1 1 0 3960.385293 -0.0277813 0.938035361
1.1.2 1440 40450 80 1200 1741 1505 105 150 311 88 71 0 1203 1338 998 106 129 197 100 84 0 0.998 1.328 1.165 1.117 1.954 1.571 0.586 52 332 2192 2868 -0.003 1095 1097 1636 1232 0 1 1 0 1208.127977 -0.0084983 0.980623358
1.1.20 5050 40440 80 18615 18363 9236 128 131 30 100 100 0 12217 12473 5620 118 122 25 100 100 0 1.528 1.476 1.425 1.382 1.573 1.546 0.92 52 320 30586 30590 0.612 18487 12099 18235 12355 0 1 1 0 13465.90077 0.0373933 1.089915076
1.1.21 5240 40440 70 1793 2060 935 112 115 25 100 100 0 1550 1629 500 115 118 22 100 100 0 1.171 1.287 1.35 1.21 1.363 1.418 0.916 32 230 3116 3462 0.228 1681 1435 1948 1514 0 1 1 0 1438.528911 -0.0222073 0.950151196
1.1.3 1640 40440 90 4070 4003 2624 111 141 278 88 86 0 2780 2511 1263 107 125 176 96 90 0 1.481 1.619 1.569 1.244 2.144 1.813 0.87 52 412 6632 6296 0.567 3959 2673 3892 2404 0 1 1 0 2874.103964 0.0775683 1.195550734
1.1.4 1830 40440 80 5552 5575 2960 121 138 71 98 96 0 2416 2340 856 111 119 32 100 100 0 2.356 2.447 2.289 1.974 2.008 2.863 0.853 52 320 7736 7683 1.236 5431 2305 5454 2229 0 1 1 0 4027.585566 0.2569353 1.806902452
1.1.5 2040 40440 90 9684 9101 4973 124 173 169 96 92 0 10332 9067 3883 116 140 91 100 100 0 0.936 1.003 0.969 0.806 1.898 1.069 0.928 52 412 19776 17928 -0.096 9560 10216 8977 8951 0 1 1 0 6629.196117 -0.1304113 0.740609554
1.1.6 2230 40440 80 3297 3068 1629 115 135 91 98 96 0 2380 2109 913 113 118 24 100 100 0 1.404 1.479 1.346 1.255 1.675 1.581 0.929 52 320 5449 4949 0.489 3182 2267 2953 1996 0 1 1 0 2180.685767 0.0384333 1.09252794
1.1.7 2450 40440 90 22767 18665 10882 108 110 24 100 100 0 20319 16803 9841 112 112 14 100 100 0 1.121 1.112 1.091 1.098 1.367 1.111 0.956 52 412 42866 35248 0.165 22659 20207 18557 16691 0 1 1 0 13703.68635 -0.0856453 0.821022488
1.1.8 2640 40440 80 6808 6592 3741 116 135 144 98 98 0 6917 6169 2610 111 114 26 100 100 0 0.983 1.069 0.99 0.899 1.81 1.169 0.878 52 315 13498 12534 -0.024 6692 6806 6476 6058 0 1 1 0 4782.296319 -0.1026933 0.789418343

Total number of rows: 6101

Table truncated, full table size 1254 Kbytes.




Supplementary data files not provided
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