36
EIGIL KJELDSEN and STEEN K0LVRAA
of conserved regions in the absence of obvious cytogenetic homologies
between human and other species.
RNA expression in situ
Much work in the area of gene expression using FISH technique has been
related to tumors, particularly in endocrine systems. Other emerging areas
using in situ techniques for RNA expression are in the fields of oncogenes,
growth factors, growth factor receptors, adhesion molecules, genes conferring resistance to chemotherapy and cytokines in inflammatory disease
(for review McNicol and Farquharson 1997).
The identification of gene expression by the detection of mRNA is
usually performed where the levels of protein product are below the limits
of immunohistochemical detection. In situ demonstration of RNA expression is of great value in confirming a tumor as the source of hormone
production where high circulating hormone levels are present but where
the tumor is immunonegative. However, detection of high levels of mRNA
does not necessarily imply a high level of functional gene product.
When RNA expression is investigated in situ, it is important to preserve the RNA in tissues. Most authors stress the importance of rapid fixation because of the potential for degradation by the ubiquitous RNases.
However, recent reports have shown that RNA can be extracted from postmortem tissues and that hybridization to mRNA can be successfully detected in tissues unfixed for at least 24 h or obtained at autopsy (Gilmore
et at. 1993). The half-life of stable RNAs in tissues has been estimated to be
24 h. To check for general preservation of RNA in tissues, the use of probes
binding to housekeeping genes or of polydT probes that will hybridize
with the polyA tail of mRNAs has been used. However, it should be remembered that the binding pattern of polydT may vary between different
tissues and a specific probe signal may be demonstrated even when polydT
is negative. Moreover, when using this technique it is important to incorporate controls to assess whether the signal, represents a specific hybridization to the appropriate target sequence.
Expression studies can also be performed in such a way as to allow
direct visual evidence of gene expression from a particular chromosome.
The technique is technically demanding but refinements of the technique
may overcome the present obstacles that restrict a more general use of this
FISH variant. An example of a potential use in the future may be in relation to the diagnosis of Beckwith-Weidemann syndrome, as the pattern of
expression of H19 and IGF2 genes on the short arm of chromosome 11 in
EIGIL KJELDSEN and STEEN K0LVRAA
of conserved regions in the absence of obvious cytogenetic homologies
between human and other species.
RNA expression in situ
Much work in the area of gene expression using FISH technique has been
related to tumors, particularly in endocrine systems. Other emerging areas
using in situ techniques for RNA expression are in the fields of oncogenes,
growth factors, growth factor receptors, adhesion molecules, genes conferring resistance to chemotherapy and cytokines in inflammatory disease
(for review McNicol and Farquharson 1997).
The identification of gene expression by the detection of mRNA is
usually performed where the levels of protein product are below the limits
of immunohistochemical detection. In situ demonstration of RNA expression is of great value in confirming a tumor as the source of hormone
production where high circulating hormone levels are present but where
the tumor is immunonegative. However, detection of high levels of mRNA
does not necessarily imply a high level of functional gene product.
When RNA expression is investigated in situ, it is important to preserve the RNA in tissues. Most authors stress the importance of rapid fixation because of the potential for degradation by the ubiquitous RNases.
However, recent reports have shown that RNA can be extracted from postmortem tissues and that hybridization to mRNA can be successfully detected in tissues unfixed for at least 24 h or obtained at autopsy (Gilmore
et at. 1993). The half-life of stable RNAs in tissues has been estimated to be
24 h. To check for general preservation of RNA in tissues, the use of probes
binding to housekeeping genes or of polydT probes that will hybridize
with the polyA tail of mRNAs has been used. However, it should be remembered that the binding pattern of polydT may vary between different
tissues and a specific probe signal may be demonstrated even when polydT
is negative. Moreover, when using this technique it is important to incorporate controls to assess whether the signal, represents a specific hybridization to the appropriate target sequence.
Expression studies can also be performed in such a way as to allow
direct visual evidence of gene expression from a particular chromosome.
The technique is technically demanding but refinements of the technique
may overcome the present obstacles that restrict a more general use of this
FISH variant. An example of a potential use in the future may be in relation to the diagnosis of Beckwith-Weidemann syndrome, as the pattern of
expression of H19 and IGF2 genes on the short arm of chromosome 11 in