5 Introduction to the Detection of Specific DNA and RNA Sequences
55
10. Wash once with PBS for 15 mins.
11. Visualize the HRP sites in a chromogen solution.
12. Wash 3 times with deionized, distilled water for 15 mins each
time, and dry up.
Check points for cell or tissue preparation
• We can use any kinds of specimens for in situ hybridization,
including cultured cells (Koji et al. 1989), fresh frozen tissues
(Koji and Brenner 1993), pre-fIxed tissues with some fIxative
(Goto et al. 1990; Ueki et al. 1995), paraffIn-embedded tissues
(Razzaque et al. 1994; Hakuno et al. 1996) and resin-embedded tissues (N akane et al. 1990). The most important point
in specimens which will be used for in situ hybridization of
specifIc mRNA is to assess the integrity and the hybridizability of RNA.
The methods to stain RNA with dyes in tissue sections or cells are
known; one is done with acridine orange and the other one with
methyl green/pyronin Y. Both staining methods are quantitatively reliable for the assessment of total RNA. However, they
do not provide any information on the accessibility of the
RNA to probes. In fact, intense RNA staining with these dyes
in specimens does not always mean its high hybridizability,
and the discrepancy between staining intensity and hybridizability is very common in specimens fIxed well with cross-linking
fIxatives. Again, although it has been widely accepted that the
degree of proteinase K digestion in the pretreatment step profoundly influences the signal intensity, especially in paraffIn sections of formalin-fIxed tissues, it had essentially no effect on the
total RNA staining with methyl green/pyronin Y (Yoshii et al.
1995).
For assessment ofhybridizability of tissue RNA, in situ hybridization of widely distributed and highly conserved RNA seemed
to be preferable. Consequently, we chose ribosomal RNA (rRNA)
as a candidate. rRNA is thought to be a superior reference to pactin mRNA and poly(A) because an occasional failure to detect
the p-actin mRNA may not be due to a loss of RNA from specimens. Rather it may be due to an extreme paucity of the mRNA,
and the probe to poly(A) does not contain G and C, resulting in
the unusual low Tm value of hybrids. By computer-assisted
searching of rat 28S rRNA cDNA, a segment consisting of 34 nu-
55
10. Wash once with PBS for 15 mins.
11. Visualize the HRP sites in a chromogen solution.
12. Wash 3 times with deionized, distilled water for 15 mins each
time, and dry up.
Check points for cell or tissue preparation
• We can use any kinds of specimens for in situ hybridization,
including cultured cells (Koji et al. 1989), fresh frozen tissues
(Koji and Brenner 1993), pre-fIxed tissues with some fIxative
(Goto et al. 1990; Ueki et al. 1995), paraffIn-embedded tissues
(Razzaque et al. 1994; Hakuno et al. 1996) and resin-embedded tissues (N akane et al. 1990). The most important point
in specimens which will be used for in situ hybridization of
specifIc mRNA is to assess the integrity and the hybridizability of RNA.
The methods to stain RNA with dyes in tissue sections or cells are
known; one is done with acridine orange and the other one with
methyl green/pyronin Y. Both staining methods are quantitatively reliable for the assessment of total RNA. However, they
do not provide any information on the accessibility of the
RNA to probes. In fact, intense RNA staining with these dyes
in specimens does not always mean its high hybridizability,
and the discrepancy between staining intensity and hybridizability is very common in specimens fIxed well with cross-linking
fIxatives. Again, although it has been widely accepted that the
degree of proteinase K digestion in the pretreatment step profoundly influences the signal intensity, especially in paraffIn sections of formalin-fIxed tissues, it had essentially no effect on the
total RNA staining with methyl green/pyronin Y (Yoshii et al.
1995).
For assessment ofhybridizability of tissue RNA, in situ hybridization of widely distributed and highly conserved RNA seemed
to be preferable. Consequently, we chose ribosomal RNA (rRNA)
as a candidate. rRNA is thought to be a superior reference to pactin mRNA and poly(A) because an occasional failure to detect
the p-actin mRNA may not be due to a loss of RNA from specimens. Rather it may be due to an extreme paucity of the mRNA,
and the probe to poly(A) does not contain G and C, resulting in
the unusual low Tm value of hybrids. By computer-assisted
searching of rat 28S rRNA cDNA, a segment consisting of 34 nu-