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

RT In Situ PCR 451
coverslips anchored with nail polish, and polyethylene bags, cut to size and held in place
with nail polish (5,17). It is important to remove ALL visible air bubbles because these will
be expanded as a result of the cycling process, which can result in some parts of sections
being inaccessible to the PCR reagents, resulting in false negatives. Different means of
covering the cell spots will result in different volumes of reagent required for each step,
and they should be adjusted accordingly to save costs.
5. Protease digestion reduces the protein cross-links that form during the fixation step, thus
allowing reagents to enter the cell. We currently use pepsin (5000 U/mL) at 37°C. The
duration of pepsin digestion depends on the type of fixative used and the extent of fixation,
with mast cell, macrophage, and lymphocyte cell lines taking ~45 min of digestion,
whereas other cells such as in vivo derived mast cells requiring much less digestion
(~15 min). However, other temperatures (22°C) and enzymes (trypsin, proteinase K) have
been used by various groups (7). Both these enzymes can give excellent results as well.
We have settled on pepsin as our enzyme of choice, as it works at low pH (3.0), and is thus
easily inactivated by a wash in water (~pH 7.5).
6. As a first step in the process of optimizing the digestion time and to become familiar with
the methodology, we use a shortened protocol (5). This involves using a single slide, with
each individual spot being subjected to a different digestion time (e.g., 20 min, 40 and
60 min). After washing off the protease, the slide is then subjected to PCR and development,
with no DNase or RT steps. By looking at the nuclear staining obtained with the different
times, the investigator will be able to pick the time that gives >90% of the nuclei positive
but maintains the cellular architecture. This optimal time can then be used for complete RT
in situ PCR studies on this same batch of fixed cells. Some investigators have noted that they
use >50% of the nuclei positive as an indication of optimal digestion (4,5). We generally see
>90% positive nuclei and have chosen this as our optimal indicator of digestion.
7. Recommended primer lengths are between 20 to 30 nucleotides. The PCR product should be
at least 300 bp in length to assure that the amplicons remain inside the cell. The RT solution
should contain an antisense primer capable of initiating first strand cDNA production.
Oligo-dT can be used, although we have found that a gene specific primer works best.
8. Numerous RT enzymes are commercially available. We have used both Maloney Murine
Leukemia Virus (MMLV) and Avian Myeloblastosis Virus (AMV) RT enzymes with
identical results. We do our RT steps at 37°C, and vary the time of incubation from 1 to 3
h, depending on the signal seen with a specific cell type.
9. The critical parameter in the composition of the PCR solution is the MgCl 2 concentration.
We found that Mg 2+ concentration in the range of 3.0 to 5.0 mM works best, which is about
four times higher than that used in solution-based PCR. This necessary increase in Mg 2+
concentration is thought to be caused by binding of Mg 2+ to the glass slides (5,7).
10. If no product is detected with the cycling program given, then the program will have to
be optimized. Take into account the T m of the primers being used, and vary the number of
cycles. In general terms, we begin with an annealing temperature that is 5°C below that of
the primer Tm. Furthermore, depending on the abundance of the mRNA being amplified,
upwards of 35 cycles may be required to obtain a detectable signal.
11. Transposed onto the technique of in situ RT-PCR is the necessity to detect the amplified
product using immunohistochemical techniques. This introduces numerous other variables
into the protocol. We have found that antibody concentrations of 0.75 to 3.75 µg/mL
provide optimal signal with little background staining.
Acknowledgments
The authors recognize Dr. Osamu Nohara for his contribution to the development of
our understanding of this procedure.