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Sample GSM244661 Query DataSets for GSM244661
Status Public on Jan 01, 2008
Title Gene expression profile of the GidA mutant-8
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 -0.81 2785 7906 120 88 220 8 3 7398 7322 2320.4 165 208 116.59 100 100 0 7233 7157 63.45 15351 15165 4882.96 145 165 62.09 100 100 0 15206 15020 247.23 2.1 2.1 2.1 2.15 0.64 1.82 0.75 1.07 22439 22177 7398 7322 7233 7157 12889 12753 12688 12552 1.75 1.75 1.75 1.8 1.53 0.81 0.81
2 0.1 3015 7906 120 88 220 8 3 2121 2266 920.68 155 204 116.01 100 100 0 1966 2111 18.28 2266 2283 826.7 132 149 47.26 100 100 0 2134 2151 47.95 1.09 1.02 1.07 1.12 0.61 0.6 0.45 0.12 4100 4262 2121 2266 1966 2111 2026 2106 1832 1919 0.93 0.87 0.92 1 0.54 -0.1 -0.1
3 -0.02 3240 7906 110 78 220 12 3 17214 17977 5473.04 163 211 122.85 100 100 0 17051 17814 140.12 21067 20731 5777.6 140 163 62.38 100 100 0 20927 20591 337.7 1.23 1.16 1.17 1.24 0.58 0.79 0.56 0.3 37978 38405 17214 17977 17051 17814 17429 17109 17232 16894 1.01 0.95 0.96 1.01 0.66 0.02 0.02
4 0.06 3445 7906 120 88 220 8 3 15955 16002 5510.68 171 223 131.76 100 100 0 15784 15831 121.09 18516 18779 6731.98 141 171 69.47 100 100 0 18375 18638 266.54 1.16 1.18 1.15 1.22 0.34 1.04 0.72 0.22 34159 34469 15955 16002 15784 15831 15352 15546 15162 15339 0.96 0.97 0.95 1 0.86 -0.06 -0.06
5 0.26 3665 7906 120 88 220 8 3 15032 14655 4800.02 179 221 118.74 100 100 0 14853 14476 126.59 15164 14928 4812.4 144 163 69.3 100 100 0 15020 14784 218.83 1.01 1.02 1.03 1.12 0.78 0.84 0.71 0.02 29873 29260 15032 14655 14853 14476 12609 12414 12427 12222 0.84 0.84 0.85 0.93 0.7 -0.26 -0.26
6 -0.01 3885 7906 120 88 220 8 3 5709 5764 2086.44 168 214 122.01 100 100 0 5541 5596 46.79 6670 6978 2848.01 132 160 62.83 100 100 0 6538 6846 106.16 1.18 1.22 1.19 1.26 0.38 1.02 0.56 0.24 12079 12442 5709 5764 5541 5596 5746 6047 5568 5875 1 1.04 1.02 1.08 0.88 0.01 0.01
7 0.88 4100 7906 110 78 220 11 3 9384 9396 2522.37 146 198 129.03 100 100 0 9238 9250 72.73 6064 6100 1733.29 131 161 71.58 100 100 0 5933 5969 84.71 0.64 0.65 0.64 0.66 0.15 0.56 0.65 -0.64 15171 15219 9384 9396 9238 9250 5179 5228 5023 5072 0.54 0.55 0.55 0.56 0.47 -0.88 -0.88
8 0.33 4325 7906 120 88 220 8 3 41662 40870 13140.62 190 253 168.39 100 100 1.1 41472 40680 247.42 40842 40488 12498.72 142 168 75.58 100 100 0 40700 40346 540.36 0.98 0.99 0.99 1.02 0.24 0.81 0.72 -0.03 82172 81026 41662 40870 41472 40680 33226 32733 32987 32426 0.8 0.8 0.8 0.82 0.66 -0.33 -0.33
9 0.07 4535 7911 120 96 220 18 3 4491 4481 1613.59 175 228 133.43 100 100 0 4316 4306 33.66 4930 4668 1687.87 141 159 56.23 100 100 0 4789 4527 87.67 1.11 1.05 1.08 1.23 1.3 0.81 0.6 0.15 9105 8833 4491 4481 4316 4306 4288 4122 4099 3941 0.95 0.9 0.92 1.07 0.71 -0.07 -0.07
10 0.61 4760 7911 130 113 220 15 3 6038 6305 2518.61 199 244 150.62 99.1 99.1 0 5839 6106 40.09 4620 4591 1844.83 153 184 88.26 100 100 0 4467 4438 52.34 0.77 0.73 0.74 0.91 1.53 0.54 0.54 -0.39 10306 10544 6038 6305 5839 6106 4003 4017 3817 3838 0.65 0.62 0.63 0.79 0.46 -0.61 -0.61
11 0.84 4970 7911 120 97 220 23 3 10085 10892 3963.7 164 229 124.09 100 100 0 9921 10728 81.27 6705 6806 2164.47 139 167 82.09 100 100 0 6566 6667 81.68 0.66 0.62 0.64 0.64 0.14 0.48 0.78 -0.6 16487 17395 10085 10892 9921 10728 5704 5783 5537 5622 0.56 0.52 0.54 0.54 0.41 -0.84 -0.84
12 0.31 5210 7906 120 96 220 6 3 14742 13874 4659.41 174 224 121.45 100 100 0 14568 13700 121.39 14368 14757 4493.48 133 161 63.08 100 100 0 14235 14624 227.79 0.98 1.07 1.01 1.14 0.37 0.82 0.72 -0.03 28803 28324 14742 13874 14568 13700 11958 12284 11786 12101 0.81 0.88 0.84 0.94 0.68 -0.31 -0.31
13 0.48 5425 7906 110 80 220 8 3 20712 20170 6034.29 164 211 117.32 100 100 0 20548 20006 176.54 18078 18713 5985.24 142 162 62.78 100 100 0 17936 18571 287.97 0.87 0.93 0.93 0.98 0.4 0.72 0.53 -0.2 38484 38577 20712 20170 20548 20006 14951 15441 14764 15234 0.72 0.76 0.76 0.8 0.6 -0.48 -0.48
14 -0.03 5640 7911 150 149 152 15 1 163 198 100.44 168 217 126.87 15.4 5.4 0 -4 30 1.28 136 162 65.82 130 148 46.02 25.5 13.4 0 6 32 2.96 -1.2 1.07 0.87 3.37 7.83 0.3 0.21 0 1 62 163 198 -4 30 165 219 6 64 -1.22 1.09 0.89 6.74 0.36 0.03 0.03
15 -0.39 5855 7906 120 88 220 16 3 6068 6013 2088.17 166 217 126.45 100 100 0 5902 5847 47.99 9270 9583 3044.74 131 150 47.07 100 100 0 9139 9452 196.95 1.55 1.62 1.61 1.75 0.76 1.11 0.58 0.63 15041 15299 6068 6013 5902 5847 7919 8211 7739 8031 1.31 1.37 1.37 1.49 0.95 0.39 0.39
16 -0.2 6095 7906 130 112 220 8 3 27115 28851 11394.19 199 245 144.82 100 100 0 26916 28652 187.23 38011 40843 14582.19 145 175 84.62 100 100 7.1 37866 40698 449.18 1.41 1.42 1.42 1.49 0.35 1.14 0.8 0.49 64782 69350 27115 28851 26916 28652 31091 33170 30848 32858 1.15 1.16 1.16 1.2 0.94 0.2 0.2
17 0.04 6310 7906 120 96 220 6 3 11571 11188 4126.15 173 222 135.03 100 100 0 11398 11015 85.69 13515 13311 4248.13 142 162 62.03 100 100 0 13373 13169 217.87 1.17 1.2 1.21 1.25 0.29 0.86 0.7 0.23 24771 24184 11571 11188 11398 11015 11290 11135 11110 10950 0.97 0.99 1.01 1.04 0.72 -0.04 -0.04
18 0.04 6525 7911 120 96 220 12 3 17143 16625 5408.29 173 232 160.92 100 100 0 16970 16452 106.53 20194 20778 6178.88 137 162 67.58 100 100 0 20057 20641 298.82 1.18 1.25 1.19 1.33 0.45 0.91 0.63 0.24 37027 37093 17143 16625 16970 16452 16715 17167 16523 16954 0.97 1.03 0.98 1.09 0.75 -0.04 -0.04
19 -0.39 6745 7911 120 96 220 12 3 33729 31978 10872.79 160 225 136 100 100 0 33569 31818 248 54418 50511 14995.02 150 177 76.81 100 100 26 54268 50361 708.51 1.62 1.58 1.58 1.76 1.06 1.03 0.56 0.69 87837 82179 33729 31978 33569 31818 44236 40850 43950 40488 1.31 1.28 1.28 1.41 0.84 0.39 0.39
20 -0.08 6965 7906 120 88 220 8 3 1505 1612 716.22 173 214 116.56 100 96.6 0 1332 1439 12.91 1770 1817 763.66 138 158 53.46 100 100 0 1632 1679 33.11 1.23 1.17 1.22 1.42 1.25 0.85 0.63 0.29 2964 3118 1505 1612 1332 1439 1613 1719 1408 1527 1.06 1.01 1.05 1.29 0.77 0.08 0.08

Total number of rows: 3392

Table truncated, full table size 925 Kbytes.




Supplementary file Size Download File type/resource
GSM244661_Cy3.tif.gz 6.3 Mb (ftp)(http) TIFF
GSM244661_Cy5.tif.gz 5.8 Mb (ftp)(http) TIFF
Processed data included within Sample table
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