Biomedical Engineering Reference
In-Depth Information
6.2.4 Reverse transcription
Primer selection, especially of the reverse primer used in the RT step, is critical since it
affects the sensitivity of the RT-PCR assay [67]. It has also been shown that tissue-specific
factors such as polysaccharides or proteins can influence amplification kinetics in a
sequence-specific manner and that this effect can be mitigated, in part, by appropriate
primer selection [68]. The structure of the RNA target at the primer-binding site must
be taken into account, as this affects the accessibility of the target to the primers. Even
so-called single-stranded RNA is typically extensively folded back onto itself, and selection
of a primer binding site in a double-stranded target site that is folded will result in a
very inefficient assay. For RNA viruses there is the additional problem of different viral
serotypes resulting in sequence variability, and it may be necessary to use a nested RT-PCR
assay with universal primers that bind to target sequences which are shared by all the
serotypes followed by a serotype-specific primer pair [69].
The most appropriate method for cDNA priming remains a contentious issue. Direct
comparison of the different methods available reveals that there is no one universally best
method, and that results are target- and enzyme-dependent [17, 18]. Random priming or
oligo-dT priming both allow a representative pool of cDNA to be produced during a single
reaction (see Protocols 6.3 and 6.4 respectively). However, it has been shown that priming
using random hexamer primers does not result in equal efficiencies of RT for all targets in
the sample and that there is not a linear correlation between input target amount and cDNA
yield when specific targets are measured [16, 70]. A recent comparison of the efficiency of
RT priming by random primers of varying lengths showed that 15-nucleotide-long random
oligonucleotides consistently yielded at least twice the amount of cDNA as random hexamers
[71]. Oligo-dT primers should only be used with intact RNA. Even when using high-quality
RNA, the cDNA molecules may be truncated, since the RT enzyme cannot proceed effi-
ciently through highly structured regions. Qiagen claim that their Omniscript (catalogue
number 205110) and Sensiscript (catalogue number 205211) reverse transcriptases can open
up and read through regions of secondary RNA structure. Nonetheless, to be on the safe
side, oligo-dT-primed assays should be targeted towards the 3
end of the transcript. This is
an unsuitable choice for experiments that require examination of splice variants, sequences
with long 3
untranslated regions, or those without polyA sequences. Furthermore, oligo-dT
priming is not recommended when using RNA extracted from paraffin tissue sections, since
formalin fixation results in the loss of the polyA tails on mRNA [72]. Target-specific primers
are the most specific and, in general, the most sensitive method for converting mRNA into
cDNA [16, 73] (see Protocol 6.5). An important argument against the use of specific priming
is that it requires a lot of target RNA and, hence, is unsuitable for the detection of numer-
ous RNA targets from limited amounts of RNA. However, a recent report demonstrates the
use of specific primers for the reliable and specific amplification of 72 genes from limiting
amounts of RNA using a multiplexed tandem (mt-) PCR approach [74]. Nevertheless, as with
random priming, there may be differences in the efficiencies with which individual RT reac-
tions occur.
