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

218 Bartlett
As with any experimental approach, the researcher must have a clear goal and
hypothesis in mind at the outset of the experimental procedure such that the techniques
selected to achieve that goal are appropriate. The attraction, and pitfall, of transcriptome
analysis is that these are extremely powerful tools for the identification of transcripts
implicated in altered tissue functions. There are therefore a few basic principles that bear
stating at this stage before the examination of the techniques available in this area.
First and most importantly, a point commonly overlooked by those researching RNA
expression is that proteins are effectors, and RNA is a blueprint. When examining RNA
expression profiles, insight is gained into the regulation of expression and stability of
the RNA species under examination, and these data must be extrapolated, usually by the
assumption that expression of RNA relates to expression of protein. This extrapolation
must be made with caution. However, the existence of the blueprint does not prove
the material for which it codes has been produced and, conversely, failure to detect
the template does not mean the protein is not present. It is therefore essential to link
RNA analyses with analyses of protein expression and function; this requirement is
frequently overlooked in transcriptome analyses.
Second, in common with all analytical techniques, it is imperative that the composition
of the tissue under examination be understood before correct interpretation of
results can be performed. Although many transcriptome analyses are performed in
monoclonal cell systems, frequently the analysis uses tissue, malignant or otherwise,
as a starting point. Where mRNA is extracted from entire tissues, there exists a strong
possibility of incorrectly assigning expression to the wrong cellular component. The
presence of blood vessels, inflammatory cells, stromal components, and a mixture of
diseased and normal tissue cells can complicate analyses. It is imperative, at some
stage of the investigation, to determine the tissue source of the mRNA detected to avoid
ascribing an incorrect origin to a protein or mRNA species.
Each approach described within this section presents specific problems that must be
sufficiently addressed to allow accurate and reliable data to be gathered if the effort
expended is to be worthwhile. However, almost all techniques aimed at global analysis
of RNA transcripts rely upon reverse transcription of mRNA, and most incorporate
polymerase chain reaction (PCR). Reverse transcription (RT) comprises the transcription
of the mRNA of choice into DNA (more accurately termed copy DNA or cDNA).
This stage of the reaction is performed using RNA virus enzymes whose role in vivo
is to transcribe viral RNA genome into a template for host transcription systems.
The earliest RT enzymes used were derived from the Molony murine leukemia virus
(Mo-MLV reverse transcriptase) and the avian myeloblastosis virus (AMV reverse
transcriptase). More recently, genetically modified forms of these enzymes with
enhanced activity have become the agents of choice. The enzymes are relatively heat
labile (particularly in comparison with Taq polymerase) and reactions are performed at
42°C with mRNA and nucleotides. The reaction must be primed because the enzymes
are dependent on double-stranded nucleic acid template. The primer is usually allowed
to anneal to the RNA after a short incubation (in the absence of enzyme) at 85°C to
denature RNA tertiary structure. Primer selection is very important. Either a primer
specific for the target sequence may be used or, more commonly, primers targeted at
copying the entire mRNA population can be used. The use of poly (dT) primers will
target mRNA poly A tails and transcribe from this point. In this case between 2 to 4 kb