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

PRINS and In Situ PCR 419
pretreatment of samples and the omission of the reverse transcription step are important
controls.
4. Conclusion
During the past 10 to 15 yr, several strategies have been developed to improve the
threshold levels of nucleic acid detection in situ by either labeling and/or amplification
of target nucleic acid sequences (prior to ISH), for example, (cycling) PRINS and in
situ PCR, or by amplification of the detection signals after the hybridization procedure,
for example, PRINS or ISH followed by tyramide signal amplification (Table 1).
Although PRINS and in situ PCR have been developed and applied in different research
disciplines and only sporadically have been compared with each other and/or with ISH
in the same study, all three techniques have been extensively studied and optimized
to cope with the central question how to achieve the most optimal balance between
specific in situ nucleic acid detection with a high signal-to-noise ratio and preservation
of cell and tissue morphology. As a result, all techniques are capable to detect repetitive
as well as single copy DNA sequences to date. However, because of evident differences
in specificity, reproducibility, and detection sensitivity between PRINS, in situ PCR,
and ISH, in our opinion the most suitable technique to be used for nucleic acid detection
in biological samples is ISH, optionally combined with tyramide signal amplification
to achieve the highest sensitivity.
In methanol:acetic acid (31)-fixed chromosome and cell preparations, a single
PRINS reaction is a suitable alternative to ISH allowing for the rapid, specific, and
reliable localization of repetitive and single copy DNA sequences (28,39). In the latter
case, PRINS should be conducted with multiple carefully selected and target-specific
oligonucleotide primers and be combined with tyramide signal amplification. The exact
detection limit, however, is unknown, and will be dependent on the ratio between the
obtained specific PRINS signal and the background noise resulting from mispriming
and labeling of nicks in the DNA, for which control experiments should be performed.
Cycling PRINS is not recommended because until now this technique has not proved
to be reliable with respect to efficiency and specificity because of problems of DNA
diffusion and nonspecific labeling of DNA (29,38,40,51). Although initially the turnaround
time (usually 16 h) and the generation and expensive purchase of probes have
been considered as real disadvantages of ISH as compared with PRINS (12), the current
situation is clearly a different one. ISH can be performed in very short turnaround times
if repetitive and directly labeled probes are used. Furthermore, ISH probes are well
available now because of the complete sequencing of the human and other genomes,
and although still expensive can be used for the specific localization of DNA probes
even under 1 kb (36) as well as for the simultaneous localization of 24 or more DNA
targets in chromosomes and cells (34,35). In contrast, in a single PRINS reaction a
huge amount of primer(s) is used together with Taq DNA polymerase and the amount
of hapten-labeled nucleotides that is normally used for labeling of 1 to 2 µg of ISH
probe. In addition, the use of tyramide signal amplification required for the localization
of single copy genes will lead to high costs if applied for clinical diagnostics as a
result of the fact that it is a patented trademark. Moreover, only a limited number of
targets can be detected simultaneously by subsequent PRINS reactions, and additional