John M. S. Bartlett.pdf - Bio-Nica.info

Differential Display Techniques 219
of cDNA can be routinely copied, which may place a constraint on PCR primer design.
RNA can be transcribed with random hexanucleotide primers (more efficient than poly
dT and produce cDNAs from the entire RNA pool); however, in this case, separation of
mRNA from ribosomal and transfer RNAs is recommended as these will also be copied
diluting the target sequences (10,11). The cDNA produced is then used for multiple
PCRs using specific sequence primers. A further novel development has been the use
of rTth DNA polymerase (from the bacterium Thermus thermophilus) allowing reverse
transcription and PCR to be performed in a single reaction tube using this thermostable
DNA polymerase with RT activity. Use of higher temperatures for the RT step with
this system allows more efficient transcription, particularly from mRNAs with high
guanine-cytosine content (12,13). The quality of mRNA extracted is an important
confounder of many experimental approaches; even with conventional and proprietary
mRNA extraction techniques, some carryover of polymerase or transcriptase inhibitors
can be observed. In extreme cases, this may reduce the efficiency of the RT to an extent
that causes errors in quantification, which may bias comparisons between experimental
samples. Therefore, every effort should be made to ensure that mRNA preparations for
quantification are as pure as possible.
2. Systems for Transcriptome Analysis
2.1. Differential Display
The use of differential display technologies has rapidly expanded over the last
decade as this technique has become established as a potent tool for the simultaneous
analysis of multiple mRNA species (14). Differential display techniques have become
as varied as the questions they are used to answer; however. in general they combine
the following three separate techniques to address this single question.
1. Production of cDNA from mRNA by RT.
2. Design of arbitrary primers to allow parts of the cDNA (tags) to be amplified by PCR.
3. Use of sequencing quality resolution by acrylamide electrophoresis.
This approach builds up a fingerprint of the RNA species expressed in different
tissue or cell samples. Comparison of these fingerprints identifies those genes that are
upregulated or downregulated in different tissues. The technique is quite elegant in
both its simplicity and power. However, as with many such techniques, the secret lies
in the careful design and interpretation of the results.
The model is best suited to the study of gene regulation in tissue culture where conditions
can be varied under careful control to avoid artifacts. Even then extreme care
must be taken to ensure that results are reproducible between experiments, the control
of tissue culture conditions is in fact the most critical phase of the experiment in
the production of accurate differential display results. Use of tissue samples makes the
approach all the more complex. Although there is obvious value in investigations
seeking to identify metastasis related gene expression, care must be taken to ensure
that, for example, differences between premetastatic and postmetastatic tissues are
related to metastasis and not a consequence of altered tumor–stroma interactions, or
differences between primary tumors. In addition, extreme care must be taken to ensure
samples to be compared are treated exactly the same to avoid artifacts being introduced
during tissue storage, mRNA extraction, or subsequent amplification.