( ...)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 frequently occurring cDNAs, and an increase in rare cDNAs. This facilitates access to the group of "least abundant" mRNAs. The difference in the number of clones, which often varied by a factor of 20,000, was drastically reduced by this normalization procedure to a factor of 40.

This was achieved by the following: Given the right circumstances, denatured, single-stranded DNAs attempt to reassociate to double-stranded DNAs. Due to the likelihood of the two matching single strands of a frequently-occurring cDNA coming together in this mixture of molecules, the frequently-occurring cDNAs reassociate much more quickly than rare cDNAs. The remaining single strands can be accumulated by a subsequent selection.

Two different normalization methods using the Eppendorf Thermomixer ⁽¹⁾ are described in the following passage:

Classical Method:

A 20 g sample of a PCR-amplified cDNA library was precipitated with ethanol, dissolved in 25 l hybridizing buffer, covered with a layer of oil, denatured for five minutes at 95C and incubated for 12 hours at 68C in the Eppendorf Thermomixer for rehybridization.

After rehybridization, three different nucleic acids were present:

1. Rehybridized ds-cDNAs from highly expressed mRNAs
2. Ss-cDNAs from rare mRNAs
3. Partly rehybridized cDNAs from frequently-occurring mRNAs

Selection of the DNA single strands occurred by restriction digestion of the double-stranded cDNAs via "frequent cutter" restriction enzymes. The single-stranded DNAs were reamplified. This normalization procedure was repeated twice.

Phenol Emulsion Technology (PERT)

"Phenol Emulsion Technology" permits more efficient normalization by accelerating rehybridization conditions in the sample ⁽²⁾. The aqueous phase of the hybridization solution is reduced by adding phenol.

10 g amplified cDNA were precipitated with ethanol and resuspended in 50 l hybrizing buffer with 8% phenol. The phases were mixed by shaking vigorously until a phenol buffer emulsion was formed. This emulsion was maintained by shaking on the Eppendorf Thermomixer. The hybridization reaction was carried out for 20 hours at 37C. A chloroform/isoamyl alcohol extraction with desalination, and selection of the single-stranded DNA took place as described.

Results

The figure shows the result of an agarose gel electrophoresis, which records the changes in the cDNA distribution caused by normalization. Before normalization (lane N0), a series of bands which represent frequently-occurring cDNAs can be seen before a diffuse background of heterogeneous cDNAs. The intensity of these bands decreases after several normalization steps (N1 and N2) have been carried out; conversely, the background of "low copy" cDNAs becomes more intense. This shows the success of normalization, which can be reexamined for cDNAs of frequently-occurring and rare mRNAs with the aid of special probes.

Peter Scheinert & Hans-Joachim Schalk, - Bernhard-Nocht-Institute of Tropical Medicine, Virology Dept.; Hamburg, Germany

Literature

(1) Scheinert, P.; Behrens, B.; Kahle, D.; & Scalk, H.-J. (1996), BIOspektrum 3/96, 50-51. Reprint available: Ref. No. 59
(2) Kohne, D.; Levison, S.; & Byers, M. (1977), Biochemistry 16, 5329-5341.

Agarose gel electrophoresis of individual phases during normalization of a cDNA library.
M: Length marker
N0: Non-normalized cDNA
N1: cDNA after one normalization
N2: cDNA after two normalizations


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