5 Introduction to the Detection of Specific DNA and RNA Sequences
53
Basic factors affecting the signal detection
During our efforts to establish the nonradioactive in situ hybridization protocol for specific RNA detection, we realized that
there are several basic points to be optimized. Some of them
are related to probe nucleic acids and the others are associated
with specimens.
Check points for probes
• As probes for in situ hybridization, there are several choices;
double-stranded cDNA, single-stranded cDNA, oligo-DNA
and cRNA. Depending upon the selection of probe nucleic
acid, what kind of labels can be chosen and how the probe
nucleic acid is labeled are different. Irrespective of selected
labels or oflabeling protocols, it is essential for successful implementation of nonradioactive in situ hybridization to verify
whether the probes are optimally labeled. Too little labeling
results in a low signal, and excessive labeling will reduce the
efficiency of hybridization and increase the risk to be suffered
from the label-compound specific background (e.g., an excess
introduction of Dig to oligo-DNA results in an increase in the
hydrophobicity of probe and becomes sticky to any hydrophobic parts of tissue sections). These issues can be mostly
assessed by dot-blot hybridization(Koji and Nakane 1990),
as shown in the Chart "Procedures for dot-blot hybridization
with T-T dimerized oligo-DNA probe". Generally speaking,
when you get 1 pg-l0 pg of complementary DNA fixed
onto a nitrocellulose or nylon membrane by the colorimetric
staining, your probe will be expected to work in in situ hybridization as well.
• The other aspect that a probe should fulfil if used for in situ
hybridization is that the labeled probes must be able to penetrate into cells and tissue sections. It has been observed frequently when large-size nucleic acids (more than 500 bases)
are haptenized whether chemically or physically and no nuclease is used during the process of haptenization, the length
of the DNA stays the same and is too large to penetrate into
cell or tissue sections. The best size of probe nucleic acid varies according to the conditions of fixation (Moench et al. 1985)
and the degree of protease digestion during pretreatment
(Koji et al. 1988). With routine pretreatment protocols, a
53
Basic factors affecting the signal detection
During our efforts to establish the nonradioactive in situ hybridization protocol for specific RNA detection, we realized that
there are several basic points to be optimized. Some of them
are related to probe nucleic acids and the others are associated
with specimens.
Check points for probes
• As probes for in situ hybridization, there are several choices;
double-stranded cDNA, single-stranded cDNA, oligo-DNA
and cRNA. Depending upon the selection of probe nucleic
acid, what kind of labels can be chosen and how the probe
nucleic acid is labeled are different. Irrespective of selected
labels or oflabeling protocols, it is essential for successful implementation of nonradioactive in situ hybridization to verify
whether the probes are optimally labeled. Too little labeling
results in a low signal, and excessive labeling will reduce the
efficiency of hybridization and increase the risk to be suffered
from the label-compound specific background (e.g., an excess
introduction of Dig to oligo-DNA results in an increase in the
hydrophobicity of probe and becomes sticky to any hydrophobic parts of tissue sections). These issues can be mostly
assessed by dot-blot hybridization(Koji and Nakane 1990),
as shown in the Chart "Procedures for dot-blot hybridization
with T-T dimerized oligo-DNA probe". Generally speaking,
when you get 1 pg-l0 pg of complementary DNA fixed
onto a nitrocellulose or nylon membrane by the colorimetric
staining, your probe will be expected to work in in situ hybridization as well.
• The other aspect that a probe should fulfil if used for in situ
hybridization is that the labeled probes must be able to penetrate into cells and tissue sections. It has been observed frequently when large-size nucleic acids (more than 500 bases)
are haptenized whether chemically or physically and no nuclease is used during the process of haptenization, the length
of the DNA stays the same and is too large to penetrate into
cell or tissue sections. The best size of probe nucleic acid varies according to the conditions of fixation (Moench et al. 1985)
and the degree of protease digestion during pretreatment
(Koji et al. 1988). With routine pretreatment protocols, a