5 Introduction to the Detection of Specific DNA and RNA Sequences
57
striking method is the use of proteolytic enzymes such as proteinase K, pronase and pepsin. With any protease, optimization
of the proteolysis conditions (concentration, digestion time,
temperature, etc.) for a given tissue is prerequisite. Over-digestion is severely deteriorative to the morphology as well as the
retention of RNA. Generally, tissues fixed with cross-linking
fixatives absolutely require protease treatment, whereas those
fixed with protein-precipitating fixatives do not always need
this step. The optimal conditions of proteolysis also depend
upon the length of probe nucleic acids; longer probes require
stronger treatment (Koji et al. 1988). Even when a short DNA
(less than 50-60 bases) such as oligo-DNA is employed, protease
digestion is still often effective (Koji et al. 1989; Yoshii et al. 1995),
indicating that the protease unmasks or exposes the target
mRNAs as well as enhancing the penetration of the probe. In
addition, the necessity of protease digestion is also different depending upon what kind of preparation or what kind of tissue is
used.
Control experiments
To verify the specificity of the signals obtained by in situ hybridization, various controls are necessary. The controls usually
performed are as follows:
(a) Pretreatment with RNase and DNase. If the signals are derived from hybridization with RNA, the signals must be lost
after RNase treatment, while DNase does not affect the signals.
(b) Use of control probes. If probes bearing sequences unrelated
to the target mRNA produce no significant staining, the signals obtained with the proper probe must be the result of
specific hybridization. In the case of single-stranded probes,
sense strand probe is conventionally used as a control probe.
When synthetic oligo-DNA probes are used, nonsense
probe, which has the same base composition to the antisense
probe, but with a different sequence, will be employed.
(c) Blocking or competition experiment. If an excess amount of
non-labeled probe nucleic acid complementary to target
57
striking method is the use of proteolytic enzymes such as proteinase K, pronase and pepsin. With any protease, optimization
of the proteolysis conditions (concentration, digestion time,
temperature, etc.) for a given tissue is prerequisite. Over-digestion is severely deteriorative to the morphology as well as the
retention of RNA. Generally, tissues fixed with cross-linking
fixatives absolutely require protease treatment, whereas those
fixed with protein-precipitating fixatives do not always need
this step. The optimal conditions of proteolysis also depend
upon the length of probe nucleic acids; longer probes require
stronger treatment (Koji et al. 1988). Even when a short DNA
(less than 50-60 bases) such as oligo-DNA is employed, protease
digestion is still often effective (Koji et al. 1989; Yoshii et al. 1995),
indicating that the protease unmasks or exposes the target
mRNAs as well as enhancing the penetration of the probe. In
addition, the necessity of protease digestion is also different depending upon what kind of preparation or what kind of tissue is
used.
Control experiments
To verify the specificity of the signals obtained by in situ hybridization, various controls are necessary. The controls usually
performed are as follows:
(a) Pretreatment with RNase and DNase. If the signals are derived from hybridization with RNA, the signals must be lost
after RNase treatment, while DNase does not affect the signals.
(b) Use of control probes. If probes bearing sequences unrelated
to the target mRNA produce no significant staining, the signals obtained with the proper probe must be the result of
specific hybridization. In the case of single-stranded probes,
sense strand probe is conventionally used as a control probe.
When synthetic oligo-DNA probes are used, nonsense
probe, which has the same base composition to the antisense
probe, but with a different sequence, will be employed.
(c) Blocking or competition experiment. If an excess amount of
non-labeled probe nucleic acid complementary to target