Hybidize DNA probes to RNA target
In general, it is possible to use DNA probes for northern blots (PCR or random-primed DNA probes). It is however important to keep in mind that:
DNA probes are best detecting abundant messages, such as housekeeping genes. For detection of rare mRNAs, Roche recommends using DIG labeled RNA probes due to the stronger signals obtained and reduced nonspecific hybridization compared to DNA probes on northern blots. In general, it is best to use RNA probes whenever possible.
There are basically two possibilities for preparing DIG labeled antisense RNA probes:
1) The standard method using a cDNA clone of your gene of interest: For making antisense probes please linearize at the 5′ end of the subclone with an appropriate restriction enzyme and using the RNA polymerase promotor located at the the 3′ end.
For linearization, use restriction endonucleases that create 5`-overhanging ends.
2) Alternatively, perform in vitro transcription from PCR fragments as templates.
Some general hints for northern analyses:
Roche recommends loading the following amounts of target RNA to your gel: max. 5 μg total RNA or 500 ng mRNA for DNA probes ( DIG-DNA probe conc. in hybridization 25 ng/mL) max. 1 μg total RNA or 100 ng mRNA for RNA probes ( DIG-RNA probe conc. in hybridization 50-100 ng/mL)
Generation of DIG-labeled RNA Probes from PCR Products
It is possible to add the appropriate RNA polymerase promoter sequences directly with your PCR primers. For a detailed experimental protocol, please refer to the attached Technical Tip.
Stripping the membrane:
Please do not use the alkali stripping method for northern blots, because RNA is alkali-labile and will be destroyed.
White Bands in northern blots
Do not load more than 1 μg of total RNA in combination with RNA probes, because the DIG System is a very sensitive detection method. You can observe the positive blocking effect, resulting in white band signals on black background, when there is too much target loaded (inherent protein contaminations will then cause this”˜blocking effect ').
Use of controls in ISH
Recommendation: Performing a negative control by incubating just the substrate with the section should not cause any background problems. A further negative control is to perform the entire ISH procedure with the sense probe or no probe, in parallel to the antisense probe, and then to perform the entire detection process, including antibody binding. The results of these tests will indicate if the antisense probe used is sticky (hybridizing unspecifically).