DNA Microarrays

    Data Access
    Experimental Conditions and RNA Isolation

    Affymetrix GeneChip® Arrays

    Spotted DNA Microarrays


    Data Access

    Several of our gene expression data sets have been entered into the ASAP database, including 41 sets of Affymetrix GeneChip array data (see list) and 50 sets of calibrated spotted array data. General experimental information can be found on this page. The expression data in the database is freely available for viewing, but is unpublished unless specified, and should be considered preliminary.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 Since these data are unpublished we request that users refrain from publishing results based on these analyses without consent from the Blattner Laboratory.
    View E. coli Gene Expression Data in ASAP
    (select Escherichia coli K-12 MG1655 from the genome drop-down menu)

    About ASAP When you enter the ASAP login page, 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 must allow the browser to accept cookies.

    Paralog Table This table contains information on an ORF's similarity with other regions of the genome. This is useful in correcting false results due to cross-hybridization (see detailed description).

    Download Published Expression Data

    In response to a number of requests, the raw data (CEL files generated by Affymetrix Microarray Suite 5.0) is now available for these publications (links to directories containing zip files):

    • JB2003 Allen, et al. (2003) J Bacteriol 185(21): 6392-6399.
    • JB2004 Herring & Blattner (2004) J Bacteriol 186(20): 6714-6720.
    • JB2005 Kang, et al. (2005) J Bacteriol 187(3): 1135-1160.
    • JBC2005 Liu, et al. (2005) J Biol Chem 280(16): 15921-15927.

    Download data sets (tab-delimited text files) published before 2001:

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    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 recipes for these media 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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    Affymetrix GeneChip® Arrays

    Description 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 45°C) 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 estimated 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.

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    Spotted DNA Microarrays

    Description 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 initially identified in the E. coli K-12 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 60°C 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.

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