13 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
187
tion of RNA in the tissue, too strong fixation may cause a false
negative because of low accessibility of the probe. Therefore, a
positive control of in situ hybridization is recommended. ~-actin
mRNA or poly(A) were used in the past as a positive control for
in situ hybridization (reviewed by Koji and Nakane 1996). However, Yoshii et al. (1995) indicated that 28S ribosomal RNA
(rRNA) has some advantages over the former controls. 28S
rRNA is widely and abundantly distributed throughout the tissue and its presence in the tissues is fairly constant. Furthermore,
in situ hybridization for 28S rRNA shows hybridizability of tissue
RNA and this helps us to assess usefulness of the protocol.
Labeling oligonucleotides with digoxigenin
(standard protocol is useful)
1. Mix
- 4 III of tailing buffer (vial 1)
4 III of CoCh solution (vial 2)
7 III of distilled water
100 pmol of oligonucleotide which has specific sequences
for a certain mRN A
1 III of DIG-dUTP (vial 3)
1 III of dATP (vial 4)
1 III of terminal transferase with 50 unit/Ill (vialS)
2. Incubate for 15 mins at 37°C
3. Place the tube on ice and add 1 III glycogen solution (vial 9)
with 200 III of 0.2 M EDTA, pH 8.0 (do not use phenol/chloroform in this step).
4. Precipitate the labeled oligonucleotide with 2.5 1l14M LiCI and
75 III prechilled ethanol. Mix well and leave for at least 30 mins
at -70 D C or 2 hrs at -20°C.
5. Centrifuge at 12,000 rpm, drain the supernatant and wash the
pellet with 50 III of 70% cold ethanol. Dry and dissolve in an
appropriate volume of sterile water. Store at _20DC until use.
187
tion of RNA in the tissue, too strong fixation may cause a false
negative because of low accessibility of the probe. Therefore, a
positive control of in situ hybridization is recommended. ~-actin
mRNA or poly(A) were used in the past as a positive control for
in situ hybridization (reviewed by Koji and Nakane 1996). However, Yoshii et al. (1995) indicated that 28S ribosomal RNA
(rRNA) has some advantages over the former controls. 28S
rRNA is widely and abundantly distributed throughout the tissue and its presence in the tissues is fairly constant. Furthermore,
in situ hybridization for 28S rRNA shows hybridizability of tissue
RNA and this helps us to assess usefulness of the protocol.
Labeling oligonucleotides with digoxigenin
(standard protocol is useful)
1. Mix
- 4 III of tailing buffer (vial 1)
4 III of CoCh solution (vial 2)
7 III of distilled water
100 pmol of oligonucleotide which has specific sequences
for a certain mRN A
1 III of DIG-dUTP (vial 3)
1 III of dATP (vial 4)
1 III of terminal transferase with 50 unit/Ill (vialS)
2. Incubate for 15 mins at 37°C
3. Place the tube on ice and add 1 III glycogen solution (vial 9)
with 200 III of 0.2 M EDTA, pH 8.0 (do not use phenol/chloroform in this step).
4. Precipitate the labeled oligonucleotide with 2.5 1l14M LiCI and
75 III prechilled ethanol. Mix well and leave for at least 30 mins
at -70 D C or 2 hrs at -20°C.
5. Centrifuge at 12,000 rpm, drain the supernatant and wash the
pellet with 50 III of 70% cold ethanol. Dry and dissolve in an
appropriate volume of sterile water. Store at _20DC until use.