In situ Hybridization of Embryos with Antisense RNA Probes - Caenorhabditis Elegans: Molecular Genetics and Development - page 251

using multiple short, nonoverlapping fluorescent probes has been described ( Raj and
Tyagi, 2010; Raj et al., 2008 ). This approach permits detection of mRNAs as
diffraction-limited spots, with the advantage that it is highly sensitive, yet specific,
and permits quantification of individual transcripts. As this protocol has very
recently been described in detail ( Raj and Tyagi, 2010; Raj et al., 2008 ), we shall
instead focus here on an alternative protocol that uses a nontoxic fixative (NTF) and
less-expensive antisense RNA probes, which offers a qualitative assessment of gene
expression. For the classic C. elegans in situ hybridization protocol, readers are
referred to the original description ( Seydoux and Fire, 1995 ), updated versions of
which can be found on the WormMethods section of WormBook ( http://www.worm-
book.org/toc_wormmethods.html ) . The procedure described here can be performed
in less than 3 days and offers good sensitivity with preservation of fine structure. It
has been used to successfully detect embryonic transcripts in C. elegans and other
nematode species ( Broitman-Maduro et al., 2009; Coroian et al., 2005 ). Others
have adapted the procedure, for example, for detection of transcripts in extruded
C. elegans gonads ( Sheth et al., 2010 ).
II. Rationale
In the method described here, whole embryos are mounted on coated glass
microscope slides, fixed, and permeabilized. Antisense RNA probes are synthesized
by in vitro transcription of PCR products, and include the use of Digoxigenin-tagged
UTP. The use of RNA probes may improve sensitivity because RNA:RNA hybrids
are more stable than DNA:RNA hybrids ( Sugimoto et al., 1995 ). Following hybrid-
ization of the probe, the slides are rinsed and processed for detection of the DIG
moiety using an anti-DIG antibody conjugated to alkaline phosphatase (AP). The
bound antibodies are detected by the use of the standard AP substrates Nitro blue
tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).
Staining is observed with a microscope equipped with differential interference
contrast (DIC), and color images are acquired with a digital camera. Positive controls
are performed with known probes, or on transgene strains with known expression.
Use of a sense RNA probe, or a mutant background known to result in the absence of
the endogenous transcript, can each serve as negative controls.
III. Methods
A. Probe Design and Synthesis
Antisense RNA probes are created from in vitro transcription of short (200 bp-1.5
kbp) PCR products carrying the T7 RNA polymerase recognition sequence at one
end. The most convenient template is genomic DNA, using primers that will amplify
as high a proportion of exon-containing sequence as possible ( Fig. 1 ). Alternatively,
a cloned cDNA fragment can be used. Some may choose to also synthesize the