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Gene Expression Arrays:,,,Highly Sensitive Detection of Expression Patterns,,,with Improved Tools for Target Amplification

tissue) are required for expression profiling.

To investigate the bias of these amplification methods, we used yeast as a model eukaryotic organism due to its low level of splicing variants. The experiments included two different cell populations grown in rich and minimal media.

Material and Methods

Sample material
Saccharomyces cerevisiae was grown in YPD medium (50 g/l, rich) and SD base medium (26.7 g/l, minimal). Two percent D+glucose (carbon source) was added to both media. Cells were grown to an OD of 0.4 in a 1 : 4 dilution and collected from 40 ml of cell suspension. Total RNA isolation was performed as described in [5].

Target preparation /amplification

Direct cDNA labeling was performed according to the MWG PAN YEAST Array Application Guide. One hundred microgram of total RNA was labeled in a 40-l labeling reaction by using moloney murine leukemia virus (MMLV) reverse transcriptase and Cy3- and Cy5-dCTP (Amersham Biosciences). Linear amplification was performed with 10 g of starting total yeast RNA according to the package inserts of the cDNA Synthesis System, Microarray Target Purification Kit, and Microarray RNA Target Synthesis Kit (T7), using Cy3- or Cy5-UTP for labeling. Random PCR amplification was performed with 50 ng total yeast RNA according to the package inserts of the Microarray Target Amplification Kit, Microarray Target Purification Kit and Microarray RNA Target Synthesis Kit (T7), using Cy3 or Cy5 for labeling.


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