168
YOSHITAKA HISHIKAWA ET AL.
To verify the results obtained by in situ hybridization, various
control experiments are required.
1. Evaluation of levels of hybridizable RNAs
To evaluate the levels ofhybridizable RNAs in tissue sections,
we used 28S rRNA probe in every run. This oligo-DNA is a
sequence complementary to the 28S rRNA sequence (nucleotide nos. 2173-2206) of rat rRNA, which are found to have
100% homology with mouse, rat and human. The oligoDNA added with 3 and 2 repeats of adenine-thymine-thymine
(ATT) at the 5' and 3' ends, respectively for the thymine-thymine (T -T) dimers. In situ localization of hybridizable rRNA
for the positive control was described previously in detail
(Y oshii 1995).
2. Positive control
As a positive control, tissues and/or cells which were already
reported to express target mRNA should be used.
3. Negative control
Sense probe should be used as a negative control in every run.
Although when using oligo-DNA probes the sense strand is
utilized as the negative control probe, a careful examinsation
should be performed, because the occurrence of anti-sense
mRNA (Le. fibroblast growth factor) has been reported
(Kimelman 1989).
4. Pretreatment with RNase
To estimate possible involvement of proteins and DNA in signal information, some sections should be digested with
RNase-A (lOOflg/ml, 37°C, 1hr) before the post-fixation step.
5. Competition experiment
Some sections are hybridized with antisense probe in the presence of an excess amount of either homologous or heterologous unlabeled oligo-DNA (about 10-20 fold) to provide definitive evidence for the sequence specificity of the signal.
Comments • For each case 2 slides should be used, one for antisense and
one for sense probe to compare the staining intensity between
the sense and antisense probes. Another should be used for
28S rRNA probe which estimates the amount ofhybridizable
RNA level in every run.
YOSHITAKA HISHIKAWA ET AL.
To verify the results obtained by in situ hybridization, various
control experiments are required.
1. Evaluation of levels of hybridizable RNAs
To evaluate the levels ofhybridizable RNAs in tissue sections,
we used 28S rRNA probe in every run. This oligo-DNA is a
sequence complementary to the 28S rRNA sequence (nucleotide nos. 2173-2206) of rat rRNA, which are found to have
100% homology with mouse, rat and human. The oligoDNA added with 3 and 2 repeats of adenine-thymine-thymine
(ATT) at the 5' and 3' ends, respectively for the thymine-thymine (T -T) dimers. In situ localization of hybridizable rRNA
for the positive control was described previously in detail
(Y oshii 1995).
2. Positive control
As a positive control, tissues and/or cells which were already
reported to express target mRNA should be used.
3. Negative control
Sense probe should be used as a negative control in every run.
Although when using oligo-DNA probes the sense strand is
utilized as the negative control probe, a careful examinsation
should be performed, because the occurrence of anti-sense
mRNA (Le. fibroblast growth factor) has been reported
(Kimelman 1989).
4. Pretreatment with RNase
To estimate possible involvement of proteins and DNA in signal information, some sections should be digested with
RNase-A (lOOflg/ml, 37°C, 1hr) before the post-fixation step.
5. Competition experiment
Some sections are hybridized with antisense probe in the presence of an excess amount of either homologous or heterologous unlabeled oligo-DNA (about 10-20 fold) to provide definitive evidence for the sequence specificity of the signal.
Comments • For each case 2 slides should be used, one for antisense and
one for sense probe to compare the staining intensity between
the sense and antisense probes. Another should be used for
28S rRNA probe which estimates the amount ofhybridizable
RNA level in every run.