Chapter 9
In Situ Hybridization for RNA:
Nonradioactive Probe: ds cDNA Probe
HIROTAKA MATSUMI AND TETSU YANO
Introduction
As has been shown previously (Koji et al. 1988; 1990; 1996), nonradioactively labeled probes are widely used for in situ hybridization because they have several advantages compared to radioactive ones.
A double-stranded cDNA (ds cDNA) probe is the most conventional. Recently, the use of a single-stranded cDNA and a
cRNA probe has been introduced. A variety of nonradioactive
labels such as biotin, digoxigenin and bromodeoxyuridine are
now available.
In this protocol, the thymine-thymine (T -T) dimer method is
used for labeling of ds cDNA (Koji et al. 1990; Hakuno et al. 1996;
Matsumi et al. 1998). In the T-T dimer method, ds cDNA, which
is assumed to contain more than two adjacent thymine residues,
is directly dimerized by UV -irradiation and can be used as a haptenic probe. The detection ofT-T dimerized probe is carried out
with immunohistochemical methods, using an anti-T -T dimer
antibody.
The conditions that a probe should fulfill if used for in situ
hybridization are that the labeled probes must be able to penetrate into cells and tissues and hybridize with nucleic acids, and
that the hybrids must be detectable using a specific detection
system. The recommended size of probe is less than 200-400
bp. Therefore, a large size of ds cDNA is used after digestion
Correspondence to Hirotaka Matsumi, University of Tokyo, Department of
Obstetrics and Gynecology, Faculty of Medicine, 7-3-1, Hongo, Bunkyo-ku,
Tokyo, 113, Japan (phone +81-3-3815-5411, Ext.3407;fax +81-3-3816-2017)
In Situ Hybridization for RNA:
Nonradioactive Probe: ds cDNA Probe
HIROTAKA MATSUMI AND TETSU YANO
Introduction
As has been shown previously (Koji et al. 1988; 1990; 1996), nonradioactively labeled probes are widely used for in situ hybridization because they have several advantages compared to radioactive ones.
A double-stranded cDNA (ds cDNA) probe is the most conventional. Recently, the use of a single-stranded cDNA and a
cRNA probe has been introduced. A variety of nonradioactive
labels such as biotin, digoxigenin and bromodeoxyuridine are
now available.
In this protocol, the thymine-thymine (T -T) dimer method is
used for labeling of ds cDNA (Koji et al. 1990; Hakuno et al. 1996;
Matsumi et al. 1998). In the T-T dimer method, ds cDNA, which
is assumed to contain more than two adjacent thymine residues,
is directly dimerized by UV -irradiation and can be used as a haptenic probe. The detection ofT-T dimerized probe is carried out
with immunohistochemical methods, using an anti-T -T dimer
antibody.
The conditions that a probe should fulfill if used for in situ
hybridization are that the labeled probes must be able to penetrate into cells and tissues and hybridize with nucleic acids, and
that the hybrids must be detectable using a specific detection
system. The recommended size of probe is less than 200-400
bp. Therefore, a large size of ds cDNA is used after digestion
Correspondence to Hirotaka Matsumi, University of Tokyo, Department of
Obstetrics and Gynecology, Faculty of Medicine, 7-3-1, Hongo, Bunkyo-ku,
Tokyo, 113, Japan (phone +81-3-3815-5411, Ext.3407;fax +81-3-3816-2017)