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DNA Microarrays
Experimental Conditions and RNA Isolation Affymetrix GeneChip® Arrays Spotted DNA Microarrays 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database is freely available for viewing, but is unpublished unless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 Since these data are unpublished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. 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): Download data sets (tab-delimited text files) published before 2001: back to topExperimental 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. RNAprotect Bacteria Reagent from Qiagen is used to stop transcription and stablize RNA before harvesting cells. MasterPure™ Complete DNA and RNA Purification Kit from Epicentre, now available from Lucigen is then used to isolate total RNA. Description of the E. coli Antisense GeneChip arrays E. coli Antisense Genome Arrays from Affymetrix were used in our lab. That product has been discontinued, and the closest current product is the E. coli Genome 2.0 Array. A detailed description of it, along with documentation comparing the two arrays, can be found at ThermoFisher. 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. 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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