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Normalizing cDNA libraries using the EppendorfThermomixer

During many experiments in molecular biology, the individual mRNA expression pattern of a cell is examined in order to investigate the reaction of a cell to exogenous influences such as viral infections or medical treatment.

To allow unstable mRNA to be examined more thoroughly, it is converted to cDNA. If all isolated mRNAs of a cell are converted to cDNA, the result is referred to as a "cDNA library." The synthesis of such a cDNA library forms an important basis for a variety of experiments, since a cDNA library allows the entire expression pattern of a cell or a cell type to be recorded.

Further examination may be hindered by the fact that the number of clones of different mRNAs which are synthesized in the cell can vary greatly. This is true of the number of individual mRNAs both within one cell and between different tissue types. For example, 16% of the total number of mRNAs in chicken liver cells is made up of one single mRNA type (serum albumin). On the other hand, some mRNAs constitute a mere 0.010.55% of the overall number. However, these so-called rare or "least abundant" mRNAs make up approximately 30% of the total mRNA information on a genome.

Cloning a cDNA library leads to over-representation of clones of highly expressed genes, whereas "least abundant" mRNAs can only be examined with difficulty.

The following method allows access to these rare mRNAs. A cDNA library was synthesized with the Eppendorf Mastercycler. To further examine these cDNAs, attempts were made to align the number of copies of the various cDNAs in the library. This is referred to as "normalizing" the cDNA library. This process leads to a decrease in the number of
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