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

434 Wiedorn and Goldmann
Fig. 1. IS-PCR can be performed with indirect and direct approach. With direct IS-PCR
during amplification, unlabeled and labeled nucleotides are used in the reaction mix. Thus,
labeled nucleotides are incorporated into the PCR products. Therefore, subsequent to the
amplification, direct detection of the labeled PCR products can be performed. In contrast with
indirect IS-PCR, only unlabeled nucleotides are used with the reaction mix. Therefore, the
amplicons are unlabeled, and for detection of the PCR products subsequent to the amplification,
an ISH with a labeled internal probe (a probe that does not contain the primer sites) has to be
performed. Thus, the detection cannot be performed until after ISH is finished.
as indirect IS-PCR, although they are predominantly associated with cell suspensions
(4,22). Optimized fixation and permeabilization as well as reduction of PCR cycle
numbers to less than 30 cycles are likely to minimize but not totally exclude this
phenomenon. With RT-IS-PCR, in contrast to solution-phase PCR, an additional
problem will arise. Depending on the primers chosen, false-positive nuclear signals
may arise because usually the primers will anneal to cDNA as well as to genomic
DNA in the nucleus (which is eliminated during RNA extraction and additional DNAse
digestion for removing residual DNA in approaches using solution phase RT-PCR) and
will therefore not only generate PCR-products in the cytoplasm but also in the nucleus.
To circumvent this problem, cDNA-specific primers, which will only anneal to the
cDNA, may be designed for RT-IS-PCR (15). In addition, a DNAse pretreatment may
be performed before RT-IS-PCR to destroy the genomic DNA. However, the general
potential of this technique is controversially discussed (5,15,26).
Nonetheless, although direct IS-PCR will contain more risks for false-positive
results, both methods are described in this chapter especially because direct IS-PCR
offers a more convenient way and will yield reliable results when performed on
nonparaffin-embedded samples like cytospins.
2. Materials
1. In situ Thermal cycling machine (Hybaid AGS, Heidelberg, Germany; Shandon, Frankfurt,
Germany) (see Note 1).
2. Wash Module (Hybaid AGS, Heidelberg, Germany; Shandon, Frankfurt, Germany).
3. SuperFrostPlus slides (Menzel Gläser, Braunschweig, Germany) (see Note 2).