Chapter 10
In Situ Hybridization for RNA:
Nonradioactive Probe: ss cDNA Probe
YOSHIO KANEMITSU AND T AKEHIKO KO]I
Introduction
The use of single-stranded cDNA (ss cDNA) is more advantageous in sensitivity than that of double-stranded cDNA (ds
cDNA) or synthetic oligonucleotide as a probe for in situ hybridization (ISH) (Sugawara et al. 1990). In this chapter, we present
a simple procedure for the production of nonradioactive ss
cDNA probe by combining the two-step asymmetric PCR and
thyminse-thyminse dimer (T -T dimer) haptenization.
Theoretically, ss cDNA probes are thought to be superior to ds
cDNA probes in detection of target mRNA, because a ss cDNA
probe does not contain a complementary strand which neutralizes itself. And compared with a oligonucleotide probe, a ss
cDNA probe can cover such a long sequence that would otherwise require a number of oligonucleotides to cover it. A singlestranded RNA probe also has such merits, but a RNA probe has a
sticky nature which increases background signal, and therefore it
often requires a ribonuclease treatment to reduce it. Furthermore, in practical use, the great stability of a DNA probe makes
for easier handling and longer storage than that of a RNA probe.
In spite of the potential advantages of ss cDNA probes in ISH
as stated above, the use of ss cDNA probes was only occasional,
partly because the preparation of the probe with M13 phage vector requires great skill and much effort. However, as shown in
this chapter, the application of the PCR to the production of the
Correspondence to Yoshio Kanemitsu, Kyushu University, Department of
Physiology, Faculty of Medicine, Maidashi, Higashi-ku, Fukuoka, 812-8582,
Japan (phone +92-642-6089; fax +92-642-6093); Takehiko Koji, Nagasaki
University School of Medicine, Department of Anatomy, Sakamoto, Nagasaki, 852, Japan
In Situ Hybridization for RNA:
Nonradioactive Probe: ss cDNA Probe
YOSHIO KANEMITSU AND T AKEHIKO KO]I
Introduction
The use of single-stranded cDNA (ss cDNA) is more advantageous in sensitivity than that of double-stranded cDNA (ds
cDNA) or synthetic oligonucleotide as a probe for in situ hybridization (ISH) (Sugawara et al. 1990). In this chapter, we present
a simple procedure for the production of nonradioactive ss
cDNA probe by combining the two-step asymmetric PCR and
thyminse-thyminse dimer (T -T dimer) haptenization.
Theoretically, ss cDNA probes are thought to be superior to ds
cDNA probes in detection of target mRNA, because a ss cDNA
probe does not contain a complementary strand which neutralizes itself. And compared with a oligonucleotide probe, a ss
cDNA probe can cover such a long sequence that would otherwise require a number of oligonucleotides to cover it. A singlestranded RNA probe also has such merits, but a RNA probe has a
sticky nature which increases background signal, and therefore it
often requires a ribonuclease treatment to reduce it. Furthermore, in practical use, the great stability of a DNA probe makes
for easier handling and longer storage than that of a RNA probe.
In spite of the potential advantages of ss cDNA probes in ISH
as stated above, the use of ss cDNA probes was only occasional,
partly because the preparation of the probe with M13 phage vector requires great skill and much effort. However, as shown in
this chapter, the application of the PCR to the production of the
Correspondence to Yoshio Kanemitsu, Kyushu University, Department of
Physiology, Faculty of Medicine, Maidashi, Higashi-ku, Fukuoka, 812-8582,
Japan (phone +92-642-6089; fax +92-642-6093); Takehiko Koji, Nagasaki
University School of Medicine, Department of Anatomy, Sakamoto, Nagasaki, 852, Japan