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The DIG System Nonradioactive and,,,Highly Sensitive Detection of Nucleic Acids

or similar applications)

Northern blotting can be performed with either DNA or RNA probes. Usually, RNA probes are more specific and more sensitive. However, if ultimate sensitivity and specificity are not required, a DNA probe may be more convenient.

If an RNA probe is to be used, the DIG Northern Starter Kit is recommended. This kit uses linearized DNA as a template and SP6, T7, or T3 RNA polymerase for the incorporation of DIG-11-UTP into the RNA transcript. Detection is performed by a chemiluminescence reaction with CDP-Star. The DIG RNA Labeling Kit contains suitable RNA expression vectors and labeling reagents.

Quantification of a labeled probe

Since the DIG System is very sensitive, it is important to work with defined amounts of probe and template to ensure optimal results. It is also important to check the efficiency of each labeling reaction by determining the amount of DIG-labeled product via spot test. This will enable you to add the correct amount of probe to the hybridization solution (Table 1).

The consequences of using the wrong amount of probe in a hybridization are obvious. Too much probe will lead to background problems; too little probe will lead to little or no hybridization signal. Table 1 lists the suggested amount of DIG-labeled probe for common hybridizations.
The benefits of the system: DIG PCR probes may be easily estimated by loading an aliquot onto an agarose gel. RNA probes may be examined for integrity (but not quantified) by gel electrophoresis.

Hybridization conditions

Optimal hybridization conditions strongly de
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