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DNA Microarrays
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Data Access
Experimental Conditions and RNA Isolation
Affymetrix GeneChip® Arrays
Spotted DNA Microarrays
List of Gene Expression Experiment
Currently there are about 50 sets of calibrated spotted array data and 41 sets of Affymetrix GeneChip array data in the ASAP database, more data will be available soon. General experimental information can be found on this page. Detailed description of each experiment can be found in the above list. Due to the large file size of the raw data files, we only provide raw data (CEL file generated by Affymetrix Microarray Suite 5.0) upon request. Please send an email to ecoli@genome.wisc.edu if you need more information.
View Gene Expression Data of E. coli
The expression data in the database is freely available for viewing but is unpublished unless specified and should be considered preliminary. The Blattner laboratory makes no claims regarding its accuracy. Since these data are unpublished we request that users refrain from publishing results based on these analyses without consent from the Blattner Laboratory.
The table above contains information on an ORF's similarity with other regions of the genome. This is useful in correcting false results due to cross-hybridization. Detailed Description
About ASAP
When you enter the ASAP login page, please login as guest and you will be able to view selected genome sequence data, annotations and gene expression data. If you would like to contribute to the information in ASAP, please contact us to become a registered user.
If you have a problem when logging in, please check the privacy settings of your bowser. In order to use ASAP, you have to allow the browser to accept cookies.
Download Expression Datasets published before 2001.
Download datasets (tab-delimited text files) published before 2001:
download
Microarray Heat Shock data
download
Microarray IPTG data
download
Radioactive Heat Shock data
download
Radioactive IPTG data
Experimental Conditions and RNA Isolation
For our experiments with microarrays, we sought a medium that was both defined and reproducible (unlike LB). Neidhardt's MOPS-based minimal medium and rich defined medium (MOPS-RDM) are now used to grow cells. The recipe for these medium are given here. RNA Bacteria Protect Reagent from Qiagen is used to stop transcription and stablize RNA before harvesting cells. MasterPure RNA isolation kit from Epicentre is then used to isolate total RNA.
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Descirption of the E.coli Antisense GeneChip arrays
E. coli antisense arrays from Affymetrix are currently used in our lab. Detailed description of it can be found at Affymetrix.
Labeling and Hybridization
Our labeling protocol is slightly different from Affymetrix's standard protocol and it can be found here. We follow the standard hybridization (16 hour at 45C) and wash protocol from Affymetrix. All chips were washed on a Fluidics Station 400, and scanned on a Hewlett-Packard (HP) GeneArray Scanner.
Data Collection and Normalization
Intensity value for each probeset is calculated by Affymetrix Microarray Suite 5.0 software uing their statistical expression algorithm. The esitmated trancription copy number (ETCN) was calculated by multiplying each signal value by an chip specific scaling factor. The scaling factor was calculated as 10,000 divided by the sum of the signal values for each protein coding gene in an experiment. The number 10,000 was chosen as an estimate for the total number of transcripts in an average cell that are expressed from protein coding genes in any given experiment.
Descirption of the E.coli Microarrays
E. coli microarrays provided by the Gene Expression Center on the UW Campus were used in our lab. These microarrays contain more than 95% of the 4,290 ORFs identified in the E. coli K12 genome as well as a variety of positive and negative controls. PCR products are double spotted onto poly-lysine coated microscope slides using standard methods.
Genomic DNA as Internal Standard -- the "Calibrator"
In order to create a database of expression profiles, we co-hybridize labeled genomic DNA and RNA then estimate the transcript abundance using the ratio of RNA signal to genomic DNA signal. The data is then scaled to allow rapid comparison of profiles obtained from different conditions.
Labeling and Hybridization
We currently generate labeled probes by reverse transcription of RNA using Cy3-dUTP and random hexamers. Genomic DNA probes are prepared by random priming using Cy5-dUTP and Klenow fragment. Probes are co-hybridized to microarrays at 60C overnight. Protocols are available here.
Data Collection and Normalization
All our spotted arrays are scanned with a SA5000 scanner from Packard Science. Quantarray® is used to extract signal intensities from the image. Data is imported into a relational database, scaled and calibrated by dividing RNA signal by genomic DNA signal.