I Subprotocol 5
Riboprobes
9 Radioactive Labeling of DNA and RNA Probes
121
Recently, methods have been developed for in vitro transcription of RNA
from cloned cDNA inserts (Krieg and Melton 1987; Melton et al. 1984).
These methods have been adapted to produce uniformly labeled, singlestranded RNA probes ("riboprobes") of high specific activity. Advantages
of such probes include the avoidance of potential reassociation of doublestranded probes (which competes with hybridization to target). Moreover,
RNA-DNA and RNA-RNA hybrids are thermodynamically more stable
than DNA-DNA hybrids permitting experimentstobe performed at higher
stringency.
For preparation of riboprobes, the template cDNA is cloned into a plasmid containing a multiple cloning site (polylinker) flanked by promoters
for two different bacteriophage RNA polymerases. For example, the multiple cloning site of the pBluescript plasmid (Stratagene) is flanked by promoters for T3 and T7 RNA polymerase. Other suitable plasmids include
pUC19, pGEM, etc. The plasmid is linearized by restriction digestion at
a site in the polylinker downstream from the cloned cDNA. Riboprobe
is then synthesized using the appropriate RNA polymerase in the presence
of three unlabeled rNTPs and one 32 P-labeled rNTP. The riboprobe may be
purified from template DNA by gel electrophoresis or deoxyribonuclease
treatment. Riboprobe prepared in this fashion is suitable for in situ hybridization, Northern or Southern blots, or nuclease protection assays.
Note: Either the coding or non-coding strand of the template cDNA will be
transcribed (depending on which ofthe two RNA polymerases is selected).
This permits generation of either "sense" or "antisense" riboprobes. Only
the latter will hybridize to the cognate mRNA. The former can be used as a
negative control, e.g., for in situ hybridization experiments.
Materials
Plasmid DNA containing the cDNA insert of interest is usually prepared on Template
silica ion-exchange columns (Qiagen) or CsCl gradients. Alkaline-lysis
"mini-prep" DNA may also be used, but it is essential to avoid contamination with ribonuclease A. Prior to use, linearize the template by restriction
digestion at a site in the polylinker downst~eam from the cloning site ( or
within the cDNA but near the distal end). Extract with phenol:chloroform:isoamyl alcohol and precipitate with ethanol. Resuspend to a concen-
Riboprobes
9 Radioactive Labeling of DNA and RNA Probes
121
Recently, methods have been developed for in vitro transcription of RNA
from cloned cDNA inserts (Krieg and Melton 1987; Melton et al. 1984).
These methods have been adapted to produce uniformly labeled, singlestranded RNA probes ("riboprobes") of high specific activity. Advantages
of such probes include the avoidance of potential reassociation of doublestranded probes (which competes with hybridization to target). Moreover,
RNA-DNA and RNA-RNA hybrids are thermodynamically more stable
than DNA-DNA hybrids permitting experimentstobe performed at higher
stringency.
For preparation of riboprobes, the template cDNA is cloned into a plasmid containing a multiple cloning site (polylinker) flanked by promoters
for two different bacteriophage RNA polymerases. For example, the multiple cloning site of the pBluescript plasmid (Stratagene) is flanked by promoters for T3 and T7 RNA polymerase. Other suitable plasmids include
pUC19, pGEM, etc. The plasmid is linearized by restriction digestion at
a site in the polylinker downstream from the cloned cDNA. Riboprobe
is then synthesized using the appropriate RNA polymerase in the presence
of three unlabeled rNTPs and one 32 P-labeled rNTP. The riboprobe may be
purified from template DNA by gel electrophoresis or deoxyribonuclease
treatment. Riboprobe prepared in this fashion is suitable for in situ hybridization, Northern or Southern blots, or nuclease protection assays.
Note: Either the coding or non-coding strand of the template cDNA will be
transcribed (depending on which ofthe two RNA polymerases is selected).
This permits generation of either "sense" or "antisense" riboprobes. Only
the latter will hybridize to the cognate mRNA. The former can be used as a
negative control, e.g., for in situ hybridization experiments.
Materials
Plasmid DNA containing the cDNA insert of interest is usually prepared on Template
silica ion-exchange columns (Qiagen) or CsCl gradients. Alkaline-lysis
"mini-prep" DNA may also be used, but it is essential to avoid contamination with ribonuclease A. Prior to use, linearize the template by restriction
digestion at a site in the polylinker downst~eam from the cloning site ( or
within the cDNA but near the distal end). Extract with phenol:chloroform:isoamyl alcohol and precipitate with ethanol. Resuspend to a concen-