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

450 Gilchrist and Befus
Fig. 3. RT in situ PCR detection of mRNA in a rat mast cell line (RCMC 1.11.2) to show
representative results. (A) Positive signal localized in the cytoplasm of RCMC indicating the
presence of TNF mRNA. (B) Positive control showing nuclear staining only and indicating that
protease digestion, PCR, and detection steps are optimized. (C) Negative control showing no
staining, indicating that genomic DNA is not causing a nonspecific signal. Original magnification
×1000; bar = 10 µm.
(4,11). In both cases, viral nucleic acid was found to be latently expressed in many
cells, a result that was only obtainable with RT in situ PCR.
This methodology is now progressing into the area of cytokine research. Cytokine
production and regulation has been difficult to study because of their low levels of
expression and rapid turnover. Previously, our knowledge of cytokine expression and
interaction was obtained from studies performed in cell culture systems, far removed
from a true in vivo environment. With recent progresses in this method, it has become
feasible to identify the source, kinetics and tissue distribution patterns of cytokines in
situ, and their patterns of distribution in both health and disease (12,13).
The future of RT in situ PCR looks promising. Growth in the area will progress with the
ability to colocalize both mRNA signals by RT in situ PCR, combined with a technique
such as immunohistochemistry for protein detection. Such a protocol will allow researchers
the power to identify both protein and gene expression in the same cell, contributing
greatly to the understanding of molecular interactions at the cellular level.
4. Notes
1. Several methods have been used to confirm the specificity of the products generated by this
methodology (8). In our experience, the labeled PCR product can be directly isolated from
the cells on the slide itself. Once isolated the product can be confirmed either by Southern
blot analysis or by cloning and sequencing of the generated product.
2. Fixation is a critical parameter in this protocol. A wide variety of fixatives have been
used with success by other investigators, including 10% formalin, 4% paraformaldehyde,
ethanol, and methanol/acetic acid (14,15). We have used 10% neutral-buffered formalin as
our fixative of choice because of its good cell structural preservation qualities and minimal
reactivity with RNA (16), thus facilitating good cDNA amplification.
3. Because cells/sections must remain on the slide through the rigors of digestion, pretreatments,
and thermocycling, they must be placed on silane-coated slides. We use slides
available from Perkin–Elmer for our in situ studies. Other sources of slides, including
those produced “in-house” as part of a regular regime for use in immunohistochemistry
can also be used with excellent results.
4. To keep the RT-PCR master mixes from evaporating during cycling, some method of
covering the sections must be devised. We have used several techniques with similar
success, including dedicated neoprene covers produced by Perkin–Elmer, as well as glass