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Sample GSM244283 Query DataSets for GSM244283
Status Public on Jan 01, 2008
Title Gene expression profile of the GidA mutant-1
Sample type RNA
 
Channel 1
Source name Log phase HSC5
Organism Streptococcus pyogenes
Characteristics HSC5 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 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 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 21, 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 Ch2 N Log Ratio (The normalized log (base2) transformation of the ratio of the medians)
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 R²
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

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 R² 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
1 0.02 2900 8026 110 78 220 8 3 11612 11746 2949.5 175 220 139.48 100 100 0 11437 11571 83.25 14691 14562 3397.62 133 148 45.56 100 100 0 14558 14429 322.43 1.27 1.25 1.27 1.29 0.29 0.79 0.47 0.35 25995 26000 11612 11746 11437 11571 11775 11703 11606 11540 1.01 0.99 1.01 1.03 0.63 0.02
2 0.27 3125 8026 120 88 220 12 3 1721 1763 778.8 169 231 147.41 100 97.7 0 1552 1594 11.67 2339 2369 877.72 125 147 45.84 100 100 0 2214 2244 51.03 1.43 1.41 1.45 1.55 0.61 0.78 0.48 0.51 3766 3838 1721 1763 1552 1594 2084 2201 1876 2009 1.21 1.19 1.23 1.39 0.7 0.27
3 -0.19 3345 8026 120 88 220 12 3 23766 23958 8322.64 180 224 132.97 100 100 0 23586 23778 178.73 25068 27167 10186.43 136 160 109.58 100 100 0 24932 27031 228.76 1.06 1.14 1.12 1.17 0.43 0.99 0.65 0.08 48518 50809 23766 23958 23586 23778 20827 22541 20653 22390 0.88 0.94 0.93 0.97 0.82 -0.19
4 0.43 3555 8026 120 88 220 4 3 17517 17109 5822.14 197 236 133.12 100 100 0 17320 16912 131.59 28425 27805 9926.55 146 164 58.78 100 100 0 28279 27659 483.56 1.63 1.64 1.62 1.75 0.81 1.46 0.74 0.71 45599 44571 17517 17109 17320 16912 23481 22880 23284 22704 1.34 1.35 1.33 1.44 1.21 0.43
5 0.5 3775 8026 120 88 220 4 3 17302 16348 4905.73 176 238 158.45 100 100 0 17126 16172 109.19 29435 27969 6786.89 139 159 54.39 100 100 0 29296 27830 541.19 1.71 1.72 1.7 1.87 0.9 1.09 0.62 0.77 46422 44002 17302 16348 17126 16172 24332 22991 24140 22820 1.41 1.42 1.4 1.54 0.9 0.5
6 0.24 3995 8026 120 88 220 4 3 5785 5921 2250.57 176 226 133.26 100 100 0 5609 5745 43.41 8458 9045 2957.1 136 150 51.01 100 100 0 8322 8909 165.8 1.48 1.55 1.59 1.73 0.95 1.04 0.63 0.57 13931 14654 5785 5921 5609 5745 6821 7335 6613 7120 1.18 1.23 1.26 1.39 0.84 0.24
7 0.14 4215 8031 120 96 220 9 3 8596 8232 3520.12 178 244 168.25 100 99 0 8418 8054 51.09 11822 11921 4956 139 157 61.24 100 100 0 11683 11782 193.06 1.39 1.46 1.39 2.18 3.25 1.04 0.55 0.47 20101 19836 8596 8232 8418 8054 9426 9535 9244 9351 1.1 1.16 1.1 1.73 0.83 0.14
8 -0.21 4435 8031 120 96 220 9 3 38847 39634 13486.39 185 256 210.07 100 100 0 38662 39449 184.93 39283 40905 13049.14 138 157 65.55 100 100 4.2 39145 40767 599.3 1.01 1.03 1.03 1.08 0.33 0.79 0.66 0.02 77807 80216 38847 39634 38662 39449 33686 34841 33517 34734 0.87 0.88 0.88 0.92 0.68 -0.21
9 -0.35 4650 8031 120 97 220 9 3 7886 7576 2821.51 182 234 144.1 100 100 0 7704 7394 54.72 7734 7179 2470.57 137 156 54.91 100 100 0 7597 7042 140.86 0.99 0.95 0.97 1.02 0.36 0.77 0.78 -0.02 15301 14436 7886 7576 7704 7394 6216 5819 6027 5626 0.78 0.76 0.77 0.82 0.62 -0.35
10 -0.3 4875 8031 130 108 220 9 3 7989 8171 2724.81 165 220 129.28 100 100 0 7824 8006 61.79 8178 8453 2738.24 140 161 53.82 100 100 0 8038 8313 151.96 1.03 1.04 1.04 1.09 0.33 0.76 0.57 0.04 15862 16319 7989 8171 7824 8006 6565 6810 6374 6618 0.81 0.82 0.82 0.87 0.61 -0.3
11 -0.19 5090 8031 120 97 220 9 3 11105 11082 3967.25 169 227 145.11 100 100 0 10936 10913 76.53 12264 12266 4528.58 141 157 50.9 100 100 0 12123 12125 240.92 1.11 1.11 1.09 1.2 0.65 0.94 0.68 0.15 23059 23038 11105 11082 10936 10913 9792 9823 9619 9655 0.88 0.88 0.87 0.96 0.75 -0.19
12 -0.07 5315 8031 130 108 220 9 3 30111 26461 10945.58 218 290 213.37 100 100 0 29893 26243 141.12 33808 31185 10089.7 151 196 144.2 100 100 0 33657 31034 234.46 1.13 1.18 1.14 1.66 1.99 0.8 0.76 0.17 63550 57277 30111 26461 29893 26243 28589 26044 28399 25885 0.95 1 0.96 1.38 0.68 -0.07
13 0.25 5530 8031 100 69 276 9 3 26485 25966 5087.39 192 281 240.05 100 100 0 26293 25774 110.33 37251 35602 8665.88 146 213 220.46 100 100 0 37105 35456 168.97 1.41 1.38 1.39 1.38 0.21 1.43 0.71 0.5 63398 61230 26485 25966 26293 25774 31467 29827 31275 29676 1.19 1.16 1.17 1.15 1.21 0.25
14 0.06 5750 8031 150 149 152 9 1 156 210 122.05 200 240 145.58 12.8 3.4 0 -43 10 1.07 145 159 49.32 134 154 52.21 24.2 7.4 0 11 25 2.78 -0.25 2.5 0.62 2.2 6.29 0.23 0.32 0 -33 35 156 210 -43 10 184 232 11 81 -0.26 2.6 0.64 7.14 0.29 0.06
15 0.17 5970 8031 120 97 220 9 3 7095 6819 2536.42 205 250 141.62 100 100 0 6890 6614 50.1 9913 9911 3385.11 140 174 98.87 100 100 0 9773 9771 100.26 1.42 1.48 1.49 1.8 1.57 0.98 0.54 0.5 16663 16385 7095 6819 6890 6614 7947 7988 7746 7782 1.12 1.17 1.18 1.43 0.79 0.17
16 -0.24 6195 8036 140 124 220 14 3 33190 31266 14725.7 182 309 246.34 100 100 0 33008 31084 134.73 33240 34615 14384.67 154 213 166.29 100 100 1.6 33086 34461 199.9 1 1.11 1.09 1.48 1.28 0.84 0.75 0 66094 65545 33190 31266 33008 31084 28186 29131 27999 28984 0.85 0.94 0.92 1.25 0.72 -0.24
17 0.26 6410 8031 120 97 220 9 3 8049 8282 2575.33 260 1394 7196.78 66 0 0 7789 8022 1.12 11959 12352 3381.96 153 635 4746.08 96.9 82.5 0 11806 12199 2.52 1.52 1.52 1.53 1.64 0.73 1.06 0.65 0.6 19595 20221 8049 8282 7789 8022 9538 9879 9340 9683 1.2 1.2 1.21 1.3 0.84 0.26
18 0.67 6635 8036 120 88 220 14 3 19694 19359 5215.66 195 591 3698.16 98.9 97.7 0 19499 19164 5.33 37251 37105 10177.47 143 288 1749.17 100 100 0 37108 36962 21.3 1.9 1.93 1.93 1.98 0.64 1.74 0.79 0.93 56607 56126 19694 19359 19499 19164 31158 30869 30956 30703 1.59 1.61 1.61 1.65 1.45 0.67
19 0.25 6850 8036 110 78 220 14 3 43305 42729 9543.31 181 241 153.29 100 100 0 43124 42548 282.5 59018 56760 10410.49 138 155 51.19 100 100 35.9 58880 56622 1152.82 1.37 1.33 1.35 1.36 0.28 0.83 0.59 0.45 102004 99170 43305 42729 43124 42548 51565 48966 51404 48905 1.19 1.16 1.18 1.18 0.73 0.25
20 1.01 7065 8036 120 88 220 16 3 1018 1150 577.86 181 222 123.03 100 96.6 0 837 969 8.27 2042 2088 725.09 130 145 45.01 100 100 0 1912 1958 45.37 2.28 2.02 2.25 2.47 1.4 0.94 0.56 1.19 2749 2927 1018 1150 837 969 1891 2022 1688 1859 2.02 1.78 1.99 2.34 0.87 1.01

Total number of rows: 3392

Table truncated, full table size 915 Kbytes.




Supplementary file Size Download File type/resource
GSM244283_Cy3.tif.gz 6.3 Mb (ftp)(http) TIFF
GSM244283_Cy5.tif.gz 5.6 Mb (ftp)(http) TIFF
Processed data included within Sample table
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