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RT In Situ PCR 445
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Reverse Transcriptase In Situ PCR
New Methods in Cellular Interrogation
Mark Gilchrist and A. Dean Befus
1. Introduction
The advent of the reverse transcriptase polymerase chain reaction (RT-PCR)
technique represents a quantum leap in sensitivity over preceding methods of detecting
mRNA transcripts, such as Northern blotting. With the arrival of such sensitive
techniques, it has become possible to amplify RNA transcripts from very small amounts
of template nucleic acid, thus opening new avenues of research that were previously
off limits because of difficulties in obtaining adequate quantities and quality of RNA
(1,2). However, RT-PCR suffers from the same limitations as its predecessor because
the isolation of RNA necessitates the destruction of the cells/tissue involved, thus
preventing the identification of the specific cell source of the mRNA (3). Conversely,
in situ hybridization allows the specific localization of mRNA to the cells of origin,
but the methodology is much less sensitive than RT-PCR (3). A methodology that
combines the best attributes of in situ hybridization (specific cellular localization) and
RT-PCR (high sensitivity) would be desirable. RT in situ PCR provides these attributes,
allowing for the location and detection of low copy RNA species, amplified within
individual intact cells (4).
The method (Fig. 1) involves fixation of cells in suspension, followed by controlled
digestion of the crosslinked cellular proteins with a proteolytic enzyme. This allows
the entry of the RT and PCR reagents into the cell. Next, genomic DNA is removed
by a DNase digestion step, thus ensuring that only mRNA is amplified. Subsequent
steps of RT and PCR are then undertaken, using a “labeled” reporter nucleotide, which
results in its direct incorporation into the PCR product. This is followed by a detection
step that visualizes the amplified mRNA, either by chromogenic or radioactive means
(Fig. 1). The specificity of the reaction can be then established by several methods
(see Note 1).
Methods of RT in situ PCR, although sharing fundamental steps, have varied greatly
between laboratories (5–7). On the basis of our experience, RT in situ PCR seems to be
best suited for the detection of mRNA in single-cell suspensions, in which fixation and
pretreatments can be optimally controlled (8). The method outlined below is similar to
that developed by Nuovo et al. 4,5) and has been adopted and built upon through our
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
445