determine mRNA distribution at the subcellular level. Using the
TSA system, it could be demonstrated that the PLT1 and PLT3
mRNAs are equally distributed throughout the cell (Fig. 3d, h).
The main pros and cons of the different staining methods are
summarized in Fig. 1.
4 Notes
1. For manual RNA (F)ISH experiments, self-made sieves
prepared from stainless-steel mesh (mesh width of
25–100 μm) glued to reaction tube slices by heat can be used.
Cut a 1.5 or 15 mL tube in ~0.8 cm rings. Heat one ring side
until the plastic starts to melt and push it on the mesh. Control
that the plastic completely seal the ring to the mesh. Remove
the surrounding mesh. The resulting sieve can be used in the
ISH experiment after sterilization with hydrogen peroxide.
The sieves are placed in a glass petri dish or multi-well plates
and the solution is exchanged outside of the sieve(s). During
liquid exchange take care that the liquid is removed completely
before adding the new liquid.
2. Coding sequence, 5
0 and 3
0 untranslated regions can be used. If
possible select several nonoverlapping regions of the mRNAencoding DNA for probe synthesis. Hybridization with either
probe should lead to the same staining pattern. If hybridization
leads to low signal strength, the application of a combination of
different probes allows to increase antigen concentration at the
target mRNA and thereby to increase staining intensity. An
entire transcript can also be used for probe synthesis but
needs to be fragmented to ~500 nucleotides, as long probes
may have negative effects on hybridization efficiencies due to
tissue penetration problems.
3. To establish RNA ISH experiments in your lab, start with
probes that have been published and are known to detect an
already described target mRNA expression pattern in your
tissue of interest. Do not use a ubiquitously expressed gene
for a start, as its stained RNA is difficult to be distinguished
from background signals. The optimal negative control for
RNA ISH experiments is the same antisense probe applied to
mutant tissues that have no or at least a reduced specific transcript level. Potential cross hybridization of probes to related
sequences leading to distorted staining pattern can only be
identified using this control. The classic negative control—a
sense probe—can generate strong staining if part of your gene
of interest is also transcribed in antisense direction. Sense control probes are therefore not always applicable. Alternatively, a
probe of comparable length and uracil content can be used as
RNA-FISH
17
TSA system, it could be demonstrated that the PLT1 and PLT3
mRNAs are equally distributed throughout the cell (Fig. 3d, h).
The main pros and cons of the different staining methods are
summarized in Fig. 1.
4 Notes
1. For manual RNA (F)ISH experiments, self-made sieves
prepared from stainless-steel mesh (mesh width of
25–100 μm) glued to reaction tube slices by heat can be used.
Cut a 1.5 or 15 mL tube in ~0.8 cm rings. Heat one ring side
until the plastic starts to melt and push it on the mesh. Control
that the plastic completely seal the ring to the mesh. Remove
the surrounding mesh. The resulting sieve can be used in the
ISH experiment after sterilization with hydrogen peroxide.
The sieves are placed in a glass petri dish or multi-well plates
and the solution is exchanged outside of the sieve(s). During
liquid exchange take care that the liquid is removed completely
before adding the new liquid.
2. Coding sequence, 5
0 and 3
0 untranslated regions can be used. If
possible select several nonoverlapping regions of the mRNAencoding DNA for probe synthesis. Hybridization with either
probe should lead to the same staining pattern. If hybridization
leads to low signal strength, the application of a combination of
different probes allows to increase antigen concentration at the
target mRNA and thereby to increase staining intensity. An
entire transcript can also be used for probe synthesis but
needs to be fragmented to ~500 nucleotides, as long probes
may have negative effects on hybridization efficiencies due to
tissue penetration problems.
3. To establish RNA ISH experiments in your lab, start with
probes that have been published and are known to detect an
already described target mRNA expression pattern in your
tissue of interest. Do not use a ubiquitously expressed gene
for a start, as its stained RNA is difficult to be distinguished
from background signals. The optimal negative control for
RNA ISH experiments is the same antisense probe applied to
mutant tissues that have no or at least a reduced specific transcript level. Potential cross hybridization of probes to related
sequences leading to distorted staining pattern can only be
identified using this control. The classic negative control—a
sense probe—can generate strong staining if part of your gene
of interest is also transcribed in antisense direction. Sense control probes are therefore not always applicable. Alternatively, a
probe of comparable length and uracil content can be used as
RNA-FISH
17