In situ Hybridization of Embryos with Antisense RNA Probes - Caenorhabditis Elegans: Molecular Genetics and Development - page 250

Abstract
Detection of transcripts in situ is a rapid means by which gene expression can be
characterized in many systems. In the nematode, Caenorhabditis elegans, the ease
with which transgenics can be made and the general reliability of reporter fusion
expression patterns, have made this technique comparatively less popular than in
other systems. There are, however, still applications in which in situ hybridization is
desired, such as for maternally expressed genes, or in related species without estab-
lished transgene methods. The most frequently used method of in situ hybridization
uses DNA probes and formaldehyde fixation. A newer approach that permits single-
transcript detection has been reported and will not be described here (Raj and Tyagi,
2010). Rather, we describe an alternative protocol that uses RNA probes with a
different fixative. This approach has been applied to C. elegans and related nema-
todes, providing reliable, sensitive detection of endogenous transcripts.
I. Introduction
Localization of transcripts by in situ hybridization is a desirable way to determine
expression patterns, because it can detect endogenous mRNA in its natural context,
and because it is a method that, once established, can be repeated on any number of
genes by changing only the antisense probe used. In addition, regulatory mechanisms
might also be identified, such as subcellular localization of the transcripts. Molecular
approaches, such as quantitative PCR (qPCR), Northern blots, or genome-wide
approaches such as microarrays or RNA-Seq require isolation of tissues, and in most
cases, amplification of the endogenous material. In the nematode, Caenorhabditis
elegans, in situ hybridization has historically not been the method of choice for
assessing endogenous gene expression, due largely to the ease of construction of
transgenic reporter strains and the general reliability of the expression patterns
produced (see Chapter on Transgenesis in this volume). Nonetheless, there remain
instances in which in situ detection of endogenous mRNA may be desired. Reporters
may be difficult to construct for particular genes, or transgenes may not express, in
particular those activated in the C. elegans germ line and very early embryo. Other
nematode species remain refractory to transgene expression techniques, either
because the DNA becomes silenced in the soma as well as germ line, or because
the necessary reagents (e.g., specific mutant backgrounds in which to make trans-
genics) do not yet exist. Newer transgene protocols may overcome some of these
limitations ( Giordano-Santini et al., 2010; Praitis et al., 2001; Schlager et al., 2009;
Semple et al., 2010 ); however, it remains to be seen whether transgenes in other
nematode species will in general be as reliable as those seen in C. elegans.
Historically, in situ detection of mRNA has relied on detection of colorimetric or
fluorescent signals from localized antisense DNA probes in whole-mount embryos
( Seydoux and Fire, 1995; Tabara et al., 1996 ). For low-abundance transcripts, signal
amplification can be used ( Bobrow and Moen, 2001 ). Recently, a new approach