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Direct and Indirect In Situ PCR 433
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Direct and Indirect In Situ PCR
Klaus Hermann Wiedorn and Torsten Goldmann
1. Introduction
In recent years, the development of in situ technologies has made good progress.
In situ hybridization (ISH) has become an important tool and has enabled the pathologist
to demonstrate infectious pathogens or mRNAs in tissue sections or cytospins without
destruction of morphology, thus enabling the assignment of signals to individual cells
or cell compartments (1–9).
Although ISH has contributed substantially to the diagnosis and understanding of
neoplastic and infectious diseases, the detection of low copy DNA and RNA sequences
by conventional ISH remained difficult in the past because of the relatively low
sensitivity of ISH, irrespective of whether the investigation was performed using
radioactive or nonradioactive hybridization probes (8,9a,10–15). Nonradioactive
probes, especially biotin and digoxigenin (DIG), which are less hazardous to work
with, can be much more quickly developed, allow a much higher spatial resolution,
and have been shown to be at least as sensitive as radioactive probes (10–14). Several
different detection systems (14,16–20) have been used to enhance signal intensities.
Nevertheless, conventional ISH usually will enable detection of high to medium copy
number nucleic acids only.
However, target amplification by in situ PCR (IS-PCR) or reverse transcription
in situ PCR (RT-IS-PCR) have been shown to even allow the detection of low copy
number DNAs or RNAs (1,4–6,8,15,21–23). There exist two different approaches to
IS-PCR: direct and indirect IS-PCR (Fig. 1).
Indirect IS-PCR requires an additional ISH step and is more cumbersome but will
usually yield reliable results. Direct IS-PCR is often hampered by nonspecific products,
especially when performed on paraffin-embedded tissue sections. These false positives
in direct IS-PCR are predominantly primer-independent artifacts resulting from DNA
repair and endogenous priming (5,21,22,24,25). These pathways are also operative in
indirect IS-PCR but will not produce false positives because no labeled nucleotides are
incorporated during the amplification step. Primer-dependent artifacts like mispriming
can be controlled by hot start maneuvers, although this is in general not quite as easy
as in solution-phase PCRs. In addition, diffusion artifacts may be involved in the
generation of false-positive cells in paraffin-embedded tissues undergoing direct as well
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
433