Chapter 14
In Situ Hybridization for RNA: Nonradioactive
Probe: RNA Probe
HARUO OHTANI
Introduction
Non -radioactive in situ hybridization method is now widely used
to detect nucleic acid in tissue sections. However, the results for
the detection of tissue mRNA are not always consistent. We have
to consider several factors for this purpose. We describe our current methods for the most consistent detection.
Procedure
Preparation of template DNA
1. The vector should contain promoter regions for RNA polymerases (for example, SP6 + T7 or T3 + T7).
2. The plasmid containing cDNA should be linealized with an
appropriate restriction enzyme, and should be purified by
ethanol precipitation.
3. The procedures should be RNase-free after phenol extraction.
In vitro transcription
Transcribe digoxigenin-labeled cRNAs of both anti-sense and
sense orientations in vitro as follows.
Haruo Ohtani, Tohoku University School of Medicine, Department of
Pathology, 2-1 Seiryo-machi, Aoba-ward, Sendai, 980-8575, Japan
In Situ Hybridization for RNA: Nonradioactive
Probe: RNA Probe
HARUO OHTANI
Introduction
Non -radioactive in situ hybridization method is now widely used
to detect nucleic acid in tissue sections. However, the results for
the detection of tissue mRNA are not always consistent. We have
to consider several factors for this purpose. We describe our current methods for the most consistent detection.
Procedure
Preparation of template DNA
1. The vector should contain promoter regions for RNA polymerases (for example, SP6 + T7 or T3 + T7).
2. The plasmid containing cDNA should be linealized with an
appropriate restriction enzyme, and should be purified by
ethanol precipitation.
3. The procedures should be RNase-free after phenol extraction.
In vitro transcription
Transcribe digoxigenin-labeled cRNAs of both anti-sense and
sense orientations in vitro as follows.
Haruo Ohtani, Tohoku University School of Medicine, Department of
Pathology, 2-1 Seiryo-machi, Aoba-ward, Sendai, 980-8575, Japan