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

PRINS and In Situ PCR 417
detection of labeled nucleotides within target or probe DNA can be performed as
described previously (Subheading 2.1. and refs. 2,46). Because of the autofluorescence
often present in tissue preparations, the limited stability of fluorochromes, and the
preference of histopathologists to analyze permanent preparations by brightfield
microscopy, colorimetric detection systems have been most frequently used (5,9). In
these procedures, the activity of alkaline phosphatase or horseradish peroxidase, coupled
to (immuno)histochemical reagents (e.g., antibodies or (strept)avidin molecules), is
visualized by enzyme precipitation reactions (NBT/BCIP and DAB are most often used,
respectively). Strikingly, in most cases suboptimal detection formats have been used,
combining, for example, only a single antibody layer (alkaline phosphatase-conjugated
anti-digoxigenin Fab fragments) with a relatively poor localizing enzyme reaction
(NBT/BCIP) and omitting the possibility to apply the sensitive tyramide signal
amplification procedure (2,14). As a consequence, relatively poor signal-to-noise ratios
have often been obtained, which may further contribute to the restricted increase in
detection sensitivity obtained by (in)direct in situ PCR when compared with ISH.
Moreover, this might also explain in part the relatively low sensitivity reported with
the ISH procedure when the PCR step is omitted, resulting in a detection limit of only
20 to 40 viral copies per cell (4,5). As can be seen in Fig. 2E, current optimal ISH
protocols (44) are able to identify the 1 to 2 copies of HPV 16 DNA integrated in the
genome in the often used SiHa control cell line without any prior PCR step.
Because the combination of PCR and ISH in the indirect in situ PCR method
is essential to guarantee that the obtained signal is specific, one may consider this
approach also as a rather cumbersome ISH method, in which sample pretreatment
consists of fixation and protease digestion in combination with heating (thermal
cycling) during nucleic acid amplification (by PCR). Moreover, the increase in detection
sensitivity as compared with conventional ISH is rather limited even after optimization
of the procedures, and cell morphology and nucleic acid localization are often poor
(see Subheading 3.4. and Table 2). Furthermore, the question arises if the increase
in the final ISH signal intensity is really caused by the in situ amplification of target
sequences alone, or is rather the result of heating the specimen by thermal cycling,
because it has been shown that microwaving or thermal cycling without in situ PCR can
also result in increased sensitivity of ISH (47,48). In this respect, we have reported a
similar effect localizing human centromere 9-specific DNA in routinely-fixed, paraffinembedded
tissue sections, where only after pepsin digestion in combination with
thermal cycling in buffer without PCR reagents it proved to be possible to perform a
primed in situ labeling reaction with a chromosome 9-specific primer with positive
outcome (Fig. 2F and ref. 14).
Indirect in situ PCR protocols are available for the simultaneous detection of two
DNA targets in formaldehyde-fixed, paraffin-embedded tissue sections as well as
for the detection of DNA in combination with a protein detected subsequently by
immunohistochemistry (49). However, without using appropriate controls (Table 3)
to adequately address the possible leakage of synthesized DNA molecules to targetnegative
sites, as is the principal drawback of indirect in situ PCR, caution is recommended
by interpretation of the data.