Biomedical Engineering Reference
In-Depth Information
sufficient RNA is available, then IVT may be preferable. On the other hand, if one has a
very small RNA sample, but is using a 3
-biased array, then PCR-based approaches may be
more appropriate. Indeed, the techniques can often be altered as well to overcome some of
the shortcomings, which are pointed out by some authors. With respect to shorter reads from
the original mRNA template, it has been shown that random priming of reverse transcrip-
tion (RT), rather than oligo-dT based priming, can also be used for PCR-based amplification
strategies, thus affording better coverage of the gene [41]. What is important to note is that
all amplification methodologies will introduce errors but that if such errors are reproducible
then they can be negated, modeled and dealt with at the informatics stage [18, 24]. In our
own investigations we have found that the various amplification methods are relatively
reproducible, and often highly reflective of a non-amplified sample (Figure 5.4).
Figure 5.4
Reproducibility and reliability of amplification strategies. HeLa and Stratagene
Universal Human Reference RNA were run on 19 000-element cDNA microarrays. 10
μ
g of each
of the RNAs was used as a control condition (gray). RNA was then amplified by T7-amplification
(blue), NuGen Ovation
™
(orange) or Global-RT-PCR (yellow). (See Plate 5.4.)
