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

440 Wiedorn and Goldmann
3.15. Primer Design
Primers should be designed to be cDNA specific (if RNA targets are under investigation),
that is, to span an intron (15) to disable amplification of genomic sequences
during RT-IS-PCR. Furthermore, primers should be chosen to give small amplification
products because the efficiency of IS-PCR will be reduced with longer products.
3.16. Closing Remarks
PCR has become an important diagnostic as well as research tool in molecular biology,
clinical chemistry, and pathology. With the invention of IS-PCR, the amplification
power of solution-phase PCR with no limitations in the amount of template was hoped
to be transferred to the in situ techniques. Unfortunately, this has become reality only
partly because IS-PCR is often hampered by poor reproducibility, specificity, and
reliability (8,9) and by the cumbersome protocol. For semiquantitative in situ studies of
gene expression in combination with image analysis, RT-IS-PCR seems to be of little
value because of the tremendously varying amplification efficiency of IS-PCR (15,27).
For these applications, ISH with subsequent signal amplification by biotinyl tyramide
proved to be the method of choice. This approach has been shown to be an excellent
alternative for IS-PCR. With respect to most applications, generally signal amplification
procedures are more suitable than target amplification by direct or indirect IS-PCR
and exhibit a sensitivity similar to that of IS-PCR (2,8,9,25,28). Adequate choice
of hybridization probes provided signal amplification allows even the detection of
single-copy virus sequences (28).
4. Notes
1. We have made good experience with the Omnislide in situ Thermocycler (Hybaid AGS
Germany) and the MISHA (Shandon Germany) because the slides are fitted horizontally
onto the blocks in the humidity chamber thus requiring sealing only during the PCR
whereas those apparatus where slides have to be fitted vertically onto the blocks require
sealing during each step of the procedure resulting in a much more cumbersome protocol.
For achieving reliable results, it is of utmost importance to use thermocyclers specially
designed for in situ PCR procedures. For detailed instructions regarding the setup of the
thermocycler and protocols, see instruction manual and (29).
2. Although expensive (E 0.50 per slide) we recommend the use of Teflon coated SuperFrost-
Plus slides (Menzel-Gläser, Braunschweig, Germany). These behaved well with respect to
adhesion of tissue even after prolonged cycling protocols and because of the hydrophobic
Teflon coating around the well do not require the cumbersome use of hydrophobic pens to
outline the reaction area. Hydrophobic pens usually have to be used several times during
a PCR protocol to guarantee a closed border around the tissue sample. However, repeated
application of the hydrophobic pens requires drying of the slide and the tissue, which can
produce strong background staining.
Slides with at least two wells should be used so that controls can be run on the same
slide simultaneously. In this case, Teflon coating will effectively prevent contamination
between the two samples. We used slides with wells of 17-mm diameter each. The volumes
indicated during the protocol proofed well in covering samples of this size but have to be
adjusted for wells with different diameters.
3. DIG-dUTP is omitted if indirect IS-PCR is performed. Instead of DIG-dUTP, biotinlabeled
nucleotides can be used too.
4. Unlike in solution-phase, PCR primers for IS-PCR should be designed to give amplicons