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Sample GSM244660 Query DataSets for GSM244660
Status Public on Jan 01, 2008
Title Gene expression profile of the GidA mutant-7
Sample type RNA
 
Channel 1
Source name Log phase TM-GidA2
Organism Streptococcus pyogenes
Characteristics A GidA mutant grown in THY media
Extracted molecule total RNA
Extraction protocol Total RNA was isolated by a protocol adapted from the method of Cheung et al. (1994) using glass beads (Lysing Matrix A, Q/BioGene) and a high-speed reciprocating shaking device (FP-120, Savant Instruments). RNA was further purified (RNeasy Mini Kit for RNA clean up, Qiagen) and contaminating DNA was removed by DNase treatments according to the manufacturers instructions (RNase free DNase set, QIAGEN and DNase I, amplification grade, Invitrogen). Representative samples were assessed for RNA integrity by electrophoretic analysis with an Agilent 2100 Bioanalyzer (Agilent Technologies) and measurement of the A260/A280 ratio was used to determine the RNA concentration and purity (accepted if >1.8).
Label Cy3
Label protocol Synthesis and labelling of cDNA used a commercial kit (3DNA Array 900, Genisphere). Briefly, cDNA was generated with random hexamers as primers for reverse transcription. The primers (1 pmole) were annealed to total RNA (4 µg) and extended with Superscript II reverse transcriptase (Invitrogen) at 42°C for 2 h. Residual RNA was then removed by alkaline treatment followed by neutralization. The efficiency of cDNA synthesis was monitored by spiking samples with a known RNA complementary to a control DNA probe spotted onto the array (Ambion). A poly T (thymine) tail was synthesized at the 3' end of the cDNA by terminal deoxynucleotidyl transferase and the capture sequence was ligated at the end of the poly T tail. The tagged cDNAs from the appropriate samples were mixed and then allowed to hybridize with the array using a commercial buffer (MWG) at 42°C overnight. Following washing using 2× SSC, 0.2% SDS, at 65°C to remove unhybridized cDNAs, the detection reagents (Cyanine 3 and Cyanine 5 3DNA dendrimers, Genisphere) were added with incubation at 65°C for 3 h. Unbound detection reagents were removed by washing.
 
Channel 2
Source name Log phase HSC5
Organism Streptococcus pyogenes
Characteristics Wild type grown in THY media
Extracted molecule total RNA
Extraction protocol Total RNA was isolated by a protocol adapted from the method of Cheung et al. (1994) using glass beads (Lysing Matrix A, Q/BioGene) and a high-speed reciprocating shaking device (FP-120, Savant Instruments). RNA was further purified (RNeasy Mini Kit for RNA clean up, Qiagen) and contaminating DNA was removed by DNase treatments according to the manufacturers instructions (RNase free DNase set, QIAGEN and DNase I, amplification grade, Invitrogen). Representative samples were assessed for RNA integrity by electrophoretic analysis with an Agilent 2100 Bioanalyzer (Agilent Technologies) and measurement of the A260/A280 ratio was used to determine the RNA concentration and purity (accepted if >1.8).
Label Cy5
Label protocol Synthesis and labelling of cDNA used a commercial kit (3DNA Array 900, Genisphere). Briefly, cDNA was generated with random hexamers as primers for reverse transcription. The primers (1 pmole) were annealed to total RNA (4 µg) and extended with Superscript II reverse transcriptase (Invitrogen) at 42°C for 2 h. Residual RNA was then removed by alkaline treatment followed by neutralization. The efficiency of cDNA synthesis was monitored by spiking samples with a known RNA complementary to a control DNA probe spotted onto the array (Ambion). A poly T (thymine) tail was synthesized at the 3' end of the cDNA by terminal deoxynucleotidyl transferase and the capture sequence was ligated at the end of the poly T tail. The tagged cDNAs from the appropriate samples were mixed and then allowed to hybridize with the array using a commercial buffer (MWG) at 42°C overnight. Following washing using 2× SSC, 0.2% SDS, at 65°C to remove unhybridized cDNAs, the detection reagents (Cyanine 3 and Cyanine 5 3DNA dendrimers, Genisphere) were added with incubation at 65°C for 3 h. Unbound detection reagents were removed by washing.
 
 
Hybridization protocol Synthesis and labelling of cDNA used a commercial kit (3DNA Array 900, Genisphere). Briefly, cDNA was generated with random hexamers as primers for reverse transcription. The primers (1 pmole) were annealed to total RNA (4 µg) and extended with Superscript II reverse transcriptase (Invitrogen) at 42°C for 2 h. Residual RNA was then removed by alkaline treatment followed by neutralization. The efficiency of cDNA synthesis was monitored by spiking samples with a known RNA complementary to a control DNA probe spotted onto the array (Ambion). A poly T (thymine) tail was synthesized at the 3' end of the cDNA by terminal deoxynucleotidyl transferase and the capture sequence was ligated at the end of the poly T tail. The tagged cDNAs from the appropriate samples were mixed and then allowed to hybridize with the array using a commercial buffer (MWG) at 42°C overnight. Following washing using 2× SSC, 0.2% SDS, at 65°C to remove unhybridized cDNAs, the detection reagents (Cyanine 3 and Cyanine 5 3DNA dendrimers, Genisphere) were added with incubation at 65°C for 3 h. Unbound detection reagents were removed by washing.
Scan protocol The signal of the bound reagents measured using a laser scanner (ScanArray Express HT, PerkinElmer).
Description An antisense oligonucleotide array based on the published genome of S. pyogenes SF370 (Ferretti et al., 2001) was made at the Microarray Core Facility in the Genome-Sequencing Center at Washington University (GSC). A single 60–80 mer oligonucleotide was designed to hybridize to the putative transcripts from each annotated open reading frame using 'TOP' software that was created at the GSC. To reduce complexity, probes for rRNA, tRNA and prophage-associated genes were not included, which resulted in an array consisting of probes for 1517 genes out of a total of 1752 predicted open reading frames in the SF370 genome. The oligonucleotides were synthesized by Illumina and were deposited onto a MWG epoxy microarray slide by custom Linear Servo Arrayer so that each slide included duplicates spots for each probe. Quality of the array was validated by hybridization with a Cyanine 5-labelled random nonamer (Hybchecker, Sigma) to ensure that the frequency of missed spots were less than 1.0% and by self-self hybridization using a single RNA sample to generate Cyanine 3- and Cyanine 5-labelled cDNAs (see below), which were then co-hybridized on the slide. Slide batches were rejected if the average ratio of hybridization differed significantly from 1.0. The purity and concentration of RNA prepared as described above was analysed by a fluorescent assay involving electrophoretic separation of RNA (Agilent 2100 bioanalyser), and rejected if the electropherogram (a diagram of fluorescence over time) showed the degradation of RNA.
Data processing The fluorescence values from each array were normalized by the LOWESS (Locally Weighted Scatter plot Smoother) function of the laser scanner. Average values were calculated from a data set consisting of 16 total microarray hybridizations that were derived from RNA samples prepared from two independent experiments, each of which was conducted on a different day. This average value represents the relative amount of cDNA binding to a given probe in competitive hybridization and is presented as a fold difference. VALUES ignored if Ch1 Median - B or Ch2 Median - B is less than 600.
 
Submission date Nov 24, 2007
Last update date Aug 14, 2011
Contact name KyuHong Cho
E-mail(s) [email protected]
Organization name Southern Illinois University Carbondale
Department Microbiology
Street address 1125 Lincoln Dr. Room 131
City Carbondale
State/province IL
ZIP/Postal code 62901
Country USA
 
Platform ID GPL4584
Series (1)
GSE9678 Gene expression of GidA mutant Streptococcus pyogenes

Data table header descriptions
ID_REF
VALUE -[INV_VALUE]
X Coordinate on x-axis of image
Y Coordinate on y-axis of image
Diameter Spot diameter in micrometers
F Pixels Total number of feature pixels
B Pixels Total number of background pixels
Footprint QC calculation (as dictated by thresholds set within quantitation parameters within ScanArray)
Flags QC calculation (as dictated by thresholds set within quantitation parameters within ScanArray)
Ch1 Median Median feature pixel intensity for ch1
Ch1 Mean Mean feature pixel intensity for ch1
Ch1 SD The standard deviation of the feature pixel intensity for ch1
Ch1 B Median Median feature background intensity for ch1
Ch1 B Mean Mean feature background intensity for ch1
Ch1 B SD The standard deviation of the feature background intensity for ch1
Ch1 % > B + 1 SD The percentage of feature pixels with intensities more than one standard deviation above the background pixel intesity for ch1
Ch1 % > B + 2 SD The percentage of feature pixels with intensities more than two standard deviations above the background pixel intesity for ch1
Ch1 F % Sat. The percentage of feature pixels for ch1 that are saturated
Ch1 Median - B The median feature pixel intensity for ch1 with the median feature background for ch1 subtracted
Ch1 Mean - B The mean feature pixel intensity for ch1 with the mean feature background for ch1 subtracted
Ch1 SignalNoiseRatio The signal-to-noise ratio for ch1, defined by (Ch1 Mean - B)/ (Ch1 B SD)
Ch2 Median Median feature pixel intensity for ch2
Ch2 Mean Mean feature pixel intensity for ch2
Ch2 SD The standard deviation of the feature pixel intensity for ch2
Ch2 B Median Median feature background intensity for ch2
Ch2 B Mean Mean feature background intensity for ch2
Ch2 B SD The standard deviation of the feature background intensity for ch2
Ch2 % > B + 1 SD The percentage of feature pixels with intensities more than one standard deviation above the background pixel intesity for ch2
Ch2 % > B + 2 SD The percentage of feature pixels with intensities more than two standard deviations above the background pixel intesity for ch2
Ch2 F % Sat. The percentage of feature pixels for ch2 that are saturated
Ch2 Median - B The median feature pixel intensity for ch2 with the median feature background for ch2 subtracted
Ch2 Mean - B The mean feature pixel intensity for ch2 with the mean feature background for ch2 subtracted
Ch2 SignalNoiseRatio The signal-to-noise ratio for ch2, defined by (Ch2 Mean - B)/ (Ch2 B SD)
Ch2 Ratio of Medians The ratio of the median intensities of each feature for each channel, with the median background subtracted
Ch2 Ratio of Means The ratio of the arithmetic mean intensities of each feature for each channel, with the median background subtracted
Ch2 Median of Ratios The median of pixel-by-pixel ratios of pixel intensities, with the median background subtracted
Ch2 Mean of Ratios The ratio of intensities, with the median background subtracted
Ch2 Ratios SD The standard deviation of pixel intensity ratios
Ch2 Rgn Ratio The regression ratio
Ch2 Rgn R_ The coefficient of determination for the current regression value
Ch2 Log Ratio The log (base2) transformation of the ratio of the medians
Sum of Medians The sum of the median intesities for each channel, with median background subtracted
Sum of Means The sum of arithmetic mean intensities for each channel, with the median background subtracted
Ch1 N Median Normalized median feature pixel intensity for ch1
Ch1 N Mean Normalized mean feature pixel intensity for ch1
Ch1 N (Median-B) The normalized median feature pixel intensity for ch1 with the median feature background for ch1 subtracted
Ch1 N (Mean-B) The normalized mean feature pixel intensity for ch1 with the mean feature background for ch1 subtracted
Ch2 N Median Normalized median feature pixel intensity for ch2
Ch2 N Mean Normalized mean feature pixel intensity for ch2
Ch2 N (Median-B) The normalized median feature pixel intensity for ch2 with the median feature background for ch2 subtracted
Ch2 N (Mean-B) The normalized mean feature pixel intensity for ch2 with the mean feature background for ch2 subtracted
Ch2 N Ratio of Medians The normalized ratio of the median intensities of each feature for each channel, with the median background subtracted
Ch2 N Ratio of Means The normalized ratio of the arithmetic mean intensities of each feature for each channel, with the median background subtracted
Ch2 N Median of Ratios The normalized median of pixel-by-pixel ratios of pixel intensities, with the median background subtracted
Ch2 N Mean of Ratios The normalized ratio of intensities, with the median background subtracted
Ch2 N Rgn Ratio The normalized regression ratio
Ch2 N Log Ratio The normalized log (base2) transformation of the ratio of the medians
INV_VALUE Ch2 N Log Ratio (The normalized log (base2) transformation of the ratio of the medians)

Data table
ID_REF VALUE X Y Diameter F Pixels B Pixels Footprint Flags Ch1 Median Ch1 Mean Ch1 SD Ch1 B Median Ch1 B Mean Ch1 B SD Ch1 % > B + 1 SD Ch1 % > B + 2 SD Ch1 F % Sat. Ch1 Median - B Ch1 Mean - B Ch1 SignalNoiseRatio Ch2 Median Ch2 Mean Ch2 SD Ch2 B Median Ch2 B Mean Ch2 B SD Ch2 % > B + 1 SD Ch2 % > B + 2 SD Ch2 F % Sat. Ch2 Median - B Ch2 Mean - B Ch2 SignalNoiseRatio Ch2 Ratio of Medians Ch2 Ratio of Means Ch2 Median of Ratios Ch2 Mean of Ratios Ch2 Ratios SD Ch2 Rgn Ratio Ch2 Rgn R_ Ch2 Log Ratio Sum of Medians Sum of Means Ch1 N Median Ch1 N Mean Ch1 N (Median-B) Ch1 N (Mean-B) Ch2 N Median Ch2 N Mean Ch2 N (Median-B) Ch2 N (Mean-B) Ch2 N Ratio of Medians Ch2 N Ratio of Means Ch2 N Median of Ratios Ch2 N Mean of Ratios Ch2 N Rgn Ratio Ch2 N Log Ratio INV_VALUE
1 -0.8 2830 8086 110 78 220 2 3 7600 7334 1815.89 158 211 136.46 100 100 0 7442 7176 55.69 14660 14567 3499.59 136 159 66.84 100 100 0 14524 14431 219.34 1.95 2.01 2.01 2.04 0.28 1.7 0.78 0.96 21966 21607 7600 7334 7442 7176 13176 13069 12996 12890 1.75 1.8 1.8 1.82 1.53 0.8 0.8
2 0.47 3055 8091 120 96 220 9 3 1909 1943 775.39 144 195 106.48 99 96.9 0 1765 1799 17.93 1518 1530 551.34 129 154 70.3 99 99 0 1389 1401 21.59 0.79 0.78 0.77 1 1.39 0.53 0.56 -0.35 3154 3200 1909 1943 1765 1799 1437 1495 1272 1337 0.72 0.71 0.7 0.95 0.5 -0.47 -0.47
3 0.11 3265 8091 120 96 220 8 3 16561 15653 6038.74 167 215 133.68 100 100 0 16394 15486 123.89 17271 16171 5262.24 140 157 55.63 100 100 0 17131 16031 310.46 1.04 1.04 1.05 1.45 3.09 0.67 0.59 0.06 33525 31517 16561 15653 16394 15486 15389 14318 15195 14131 0.93 0.92 0.93 1.28 0.6 -0.11 -0.11
4 -0.09 3490 8091 110 81 220 7 3 15983 16000 4833.86 160 206 120.95 100 100 0 15823 15840 132.14 19127 18328 5691.58 137 160 57.79 100 100 0 18990 18191 330.99 1.2 1.15 1.13 1.24 0.69 0.8 0.46 0.26 34813 34031 15983 16000 15823 15840 17035 16195 16836 16002 1.06 1.02 1 1.09 0.71 0.09 0.09
5 0.3 3715 8086 120 88 220 6 3 13669 13399 4108.99 169 215 120.29 100 100 0 13500 13230 113.63 12427 11430 4272.6 138 163 64.27 100 100 0 12289 11292 193.36 0.91 0.85 0.89 0.95 1.09 0.86 0.68 -0.14 25789 24522 13669 13399 13500 13230 11117 10193 10946 10026 0.81 0.76 0.79 0.84 0.77 -0.3 -0.3
6 0.01 3930 8091 120 97 220 7 3 5434 5192 2012.87 155 210 120.57 100 100 0 5279 5037 45.07 5914 6180 2566.37 133 154 58.18 100 100 0 5781 6047 101.65 1.1 1.2 1.16 1.27 0.57 0.99 0.6 0.13 11060 11084 5434 5192 5279 5037 5407 5692 5245 5532 0.99 1.09 1.05 1.16 0.91 -0.01 -0.01
7 0.67 4150 8091 110 81 220 7 3 9139 9160 2712.6 155 204 120.94 100 100 0 8984 9005 75.57 6386 6336 2099.36 145 167 58.85 100 100 0 6241 6191 108.51 0.69 0.69 0.69 0.74 0.41 0.55 0.51 -0.53 15225 15196 9139 9160 8984 9005 5801 5767 5642 5606 0.63 0.62 0.63 0.67 0.5 -0.67 -0.67
8 0.5 4365 8091 120 96 220 8 3 35109 36003 12126.11 186 229 136.66 100 100 0 34923 35817 256.91 28247 28418 9925.94 145 171 66.02 100 100 0 28102 28273 427.86 0.8 0.79 0.8 0.9 0.9 0.63 0.6 -0.31 63025 64090 35109 36003 34923 35817 24962 24700 24698 24451 0.71 0.69 0.7 0.78 0.56 -0.5 -0.5
9 0.03 4585 8096 120 88 220 13 3 4100 4213 1337.69 146 195 111.56 100 100 0 3954 4067 36.75 4418 4386 1520.38 151 172 68.77 100 100 0 4267 4235 64.24 1.08 1.04 1.07 1.05 0.27 0.87 0.59 0.11 8221 8302 4100 4213 3954 4067 4058 4081 3875 3901 0.98 0.95 0.97 0.96 0.8 -0.03 -0.03
10 0.07 4810 8091 120 97 220 7 3 5894 6229 2017.35 186 234 153.24 100 100 0 5708 6043 38.46 6161 6039 1739.58 149 173 76.75 100 100 0 6012 5890 80.27 1.05 0.97 1.01 1.02 0.23 0.61 0.51 0.07 11720 11933 5894 6229 5708 6043 5627 5546 5453 5376 0.96 0.88 0.91 0.93 0.56 -0.07 -0.07
11 0.86 5020 8091 120 97 220 11 3 11099 11125 3941.48 166 222 130.37 100 100 0 10933 10959 85.13 6838 7260 2652.74 135 158 64.22 100 100 0 6703 7125 106.48 0.61 0.65 0.63 0.67 0.16 0.56 0.69 -0.71 17636 18084 11099 11125 10933 10959 6189 6554 6036 6401 0.55 0.59 0.57 0.6 0.51 -0.86 -0.86
12 -0.29 5250 8091 120 97 220 7 3 12767 12096 3626.89 166 217 121.89 100 100 0 12601 11930 104.74 17490 16283 4622.94 135 156 57.6 100 100 0 17355 16148 303.63 1.38 1.35 1.36 1.43 0.48 1.02 0.64 0.46 29956 28078 12767 12096 12601 11930 15611 14467 15423 14284 1.22 1.2 1.21 1.26 0.91 0.29 0.29
13 0.14 5460 8091 110 81 220 11 3 19553 18797 5857.92 157 210 126.79 100 100 0 19396 18640 154.21 20059 19657 6596.18 142 162 61.15 100 100 0 19917 19515 328 1.03 1.05 1.05 1.16 0.81 0.89 0.63 0.04 39313 38155 19553 18797 19396 18640 17835 17316 17625 17112 0.91 0.93 0.93 1.01 0.79 -0.14 -0.14
14 -0.05 5680 8091 150 149 152 11 1 150 185 93.91 161 198 109.96 14.8 6 0 -10 24 1.36 139 154 47.68 138 159 58.8 20.1 3.4 0 1 16 2.36 -0.09 0.67 0.83 1.7 2.22 0.31 0.37 0 -10 40 150 185 -10 24 161 201 1 37 -0.09 0.69 0.86 3.95 0.36 0.05 0.05
15 -0.26 5905 8096 120 88 220 13 3 5239 5479 1923.52 167 209 123.26 100 100 0 5072 5312 42.5 6818 6708 1973.06 130 153 55.51 100 100 0 6688 6578 122.82 1.32 1.24 1.27 1.37 0.66 0.85 0.69 0.4 11760 11890 5239 5479 5072 5312 6228 6163 6066 6006 1.2 1.12 1.15 1.25 0.78 0.26 0.26
16 -0.13 6140 8091 130 113 220 12 3 18161 19743 7593.31 163 202 116.13 100 100 0 17998 19580 156.39 22398 22484 8269.22 138 159 56.57 100 100 0 22260 22346 395.94 1.24 1.14 1.17 1.19 0.39 0.89 0.66 0.31 40258 41926 18161 19743 17998 19580 19910 19757 19693 19545 1.09 1.01 1.04 1.04 0.79 0.13 0.13
17 0.45 6345 8091 120 96 220 8 3 9574 9980 3571.09 143 190 104.83 100 100 0 9431 9837 91.33 7837 8058 2630.31 147 170 68.59 100 100 0 7690 7911 114.25 0.82 0.8 0.81 0.86 0.36 0.55 0.55 -0.29 17121 17748 9574 9980 9431 9837 7095 7275 6926 7108 0.73 0.72 0.73 0.77 0.49 -0.45 -0.45
18 0 6560 8096 120 96 220 15 3 15440 15393 5197.64 164 220 132.79 100 100 0 15276 15229 116.28 17422 17598 6024.63 161 187 86.96 100 100 0 17261 17437 200.35 1.13 1.14 1.11 1.26 0.73 0.98 0.72 0.18 32537 32666 15440 15393 15276 15229 15530 15567 15318 15356 1 1.02 0.99 1.11 0.88 0 0
19 -0.29 6785 8096 120 88 220 13 3 32197 31528 9823.16 163 223 146.69 100 100 0 32034 31365 219.49 44752 46079 14232.1 154 186 91.49 100 100 11.4 44598 45925 489.13 1.39 1.46 1.49 1.52 0.44 1.22 0.71 0.48 76632 77290 32197 31528 32034 31365 39454 39874 39090 39536 1.22 1.28 1.3 1.31 1.07 0.29 0.29
20 -0.33 7005 8096 120 88 220 13 3 1362 1424 616.53 154 207 123.43 100 98.9 0 1208 1270 11.03 1791 1728 603.16 138 156 52.03 100 100 0 1653 1590 34.42 1.37 1.25 1.31 1.4 0.55 0.85 0.76 0.45 2861 2860 1362 1424 1208 1270 1702 1699 1519 1533 1.26 1.15 1.2 1.35 0.81 0.33 0.33

Total number of rows: 3392

Table truncated, full table size 928 Kbytes.




Supplementary file Size Download File type/resource
GSM244660_Cy3.tif.gz 6.2 Mb (ftp)(http) TIFF
GSM244660_Cy5.tif.gz 5.8 Mb (ftp)(http) TIFF
Processed data included within Sample table
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