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

418 Speel, Ramaekers, and Hopman
3.4 Sensitivity and Evaluation of In Situ PCR Results
Only a few studies have tried to give an indication of the increase in in situ signal
intensity by comparing indirect in situ PCR with ISH. In the most optimal situation,
a 50-fold increase in sensitivity has been reported when cell preparations were used
(7,50). This relatively poor result after PCR amplification thus lies far beneath the
amplification efficiency that can be achieved by solution-phase PCR. As has been
discussed above, this is the result of many factors, including suboptimal sample
pretreatment, inefficient and nonspecific in situ primer extension, as well as diffusion of
PCR products because of the denaturation steps during PCR. In addition, background
signals introduced by the ISH procedure, as a result of nonspecific binding of probe and
detection reagents to the sample and the coated glass slide as the result of suboptimal
stringent washings after probe hybridization and application of too less diluted probe
and/or detection conjugates, may even further contribute to a decrease of the difference
in signal intensity between in situ PCR and ISH. Because so many factors may influence
the final signal intensity observed, it is not advisable to use in situ PCR results for
quantification purposes.
Nevertheless, an amplification factor of 10- to 50-fold would still be a very acceptable
increase in sensitivity, provided that reproducible and specific results would be
obtained by indirect in situ PCR. However, despite the fact that the detection of one
target copy per cell have been reported by using a single primer pair that amplifies
a sequence of a few hundred basepairs (5,10), indirect in situ PCR appears also to
be hampered by restricted specificity of results and, moreover, is unable to distinctly
localize nucleic acid sequences in cell and tissue preparations (9,51). Even when
assuming that PCR conditions are optimal and artifacts caused by mispriming and
nicks are eliminated, the diffusion of generated PCR products from the site of synthesis
inside and/or outside the cell is an almost impossible process to control. Therefore,
many creative approaches intended to minimize the impact of diffusion have been
described, such as optimal sample fixation and pretreatment, reduction of PCR cycle
numbers, generation of longer or more complex PCR products, incorporation of labeled
nucleotides to make bulkier PCR products, and the embedding of samples in agarose
or protein matrices (9,43,50,52). Nevertheless, in most cases only a discrimination
between positive and negative nuclei can be determined, whereas for example the
number and site of viral integration in the nucleus, as shown in Fig. 2E by ISH,
as well as the discrimination between viral integration and replication, are almost
impossible to identify.
Thus, appropriate controls at each step in the (in)direct in situ PCR procedure are
essential to demonstrate specificity and to correctly interpret the results (Table 3 and
refs. 5,9,42). Control experiments should include (1) samples that either harbor or
lack the target of interest (or a known mixture of these cells to verify the expected
result) or use of irrelevant primers that cannot find targets in the cells under study (e.g.,
viral-specific primers in uninfected cells); (2) omission of the DNA polymerase from
the PCR mixture to detect nonspecific sticking of ISH probes and detection reagents
to the slide; (3) omission of primers to detect artifacts related to endogenous priming
(nicks in the DNA) and DNA repair in the direct in situ PCR approaches. In case
of reverse transcription in situ PCR to detect (m)RNA sequences in situ, RNase