Digoxigenin (DIG)-labeled, single-stranded RNA probes of defined length are generated by in vitro transcription. DIG-11-UTP is incorporated by SP6, T7, and T3 RNA polymerases at approximately every 20 to 25th nucleotide of the transcript under standard conditions. The DIG RNA Labeling Mix (Product No. 11277073910) is specifically designed for use with SP6, T7, and T3 RNA polymerases. The kit is supplied with an optimized transcription buffer and nucleotide mixture for labeling RNA with digoxigenin-11-UTP.
Determine the labeling efficiency in terms of μg (expected yield of a standard labeling reaction is 20 μg of DIG-labeled RNA per microgram of linearized template DNA after the DIG RNA labeling reaction).
For best results, load an aliquot on an agarose gel. RNA transcripts can be analyzed using agarose gel electrophoresis (e.g., formaldehyde gels or native gels) and ethidium bromide staining.
Note: RNA probes may be examined for integrity, but not quantified by gel electrophoresis.
Generating DIG-labeled RNA probe directly from a PCR product without subcloning it into an expression vector:
This is possible if the appropriate RNA polymerase promoter sequences is included directly into the PCR primers.
For a detailed experimental protocol, please read the attached Technical Tip.
Circular or nicked plasmid DNA in the transcription reaction may interfere with transcription, making it less efficient and producing non-specific transcripts. It is best to use only purified plasmid DNA to ensure that restriction enzyme digestion is complete. For best results, use the High Pure Plasmid Isolation Kit (Product No. 11754777001). Use 10 units of restriction enzyme per microgram DNA and digest for at least 3 hours. It is very important that the template is of high purity and free of contaminants such as salts, proteins, RNAse, etc. For best results, gel-purify the linearized template and use a spin column for further clean-up, for example the High Pure PCR Product Purification Kit (Product No. 11732668001). Alternatively, phenol/chloroform extraction and ethanol precipitation can also be performed. Resuspend the template DNA in RNAse-free water (Water, PCR grade), check an aliquot on a gel for size, purity and concentration and use 1 μg of template in the in vitro transcription reaction.
Possibility 1: Under nondenaturing conditions (simple agarose gel), secondary RNA structure may not be resolved resulting in two bands. To verify, use a MOPS/formaldehyde gel (as commonly used for northern gels ) or a PAGE.
Possibility 2: Template DNA used to generate RNA probes is still present in marginal amounts in the aliquot loaded onto the gel, when no DNAse digestion step was performed. When using the controls supplied in the DIG RNA Labeling Kit, please refer to the package insert for details on the DNA fragments they contain.
Possibility 3: Depending on the DNA construct used, the availability of 5 ' overhangs for linearization is recommended. After the DIG labeling reaction, check results by gel electrophoresis to make sure the transcript has the expected size. There are some DNA sequences, which may cause the RNA polymerases to produce abortive or shortened transcripts. These problems may be overcome by recloning into a vector with the opposite direction of the polylinker and transcribing the same DNA-strand with a different RNA polymerase or transcribing the other strand of the template DNA.
DNA templates with 3`-overhanging or blunt ends may in some cases yield unwanted transcripts of the "wrong" DNA strand, caused by "run on" transcription. To avoid this, use restriction endonucleases which create 5`-overhanging ends.