The first Northern blot I did in my life was using 32
P-labeled probes, and I still remember how nervous I was hearing the buzzing of the Geiger counter and how inconvenient it was to perform all of the experiments behind a shield. I was wondering if there was a non-radioactive way to probe a Northern blot. I did a Google search and found the DIG system from Roche. The DIG system allows safe and efficient labeling of DNA, RNA, or oligonucleotides, which can then be used as probes for a variety of hybridization reactions, including Northern and Southern blots, in situ
hybridization, arrays, etc. In my review, I will focus on the application of the DIG system for detection on Northern blots.
Either DNA or RNA probes can be used for detecting RNA expression via a Northern blot. Generally, RNA probes are more specific and sensitive. For RNA probe synthesis, I used T7 RNA polymerase for in vitro transcription and incorporation of Dig-UTP (cat#11175025910) into the RNA probes. After hybridization and stringency washes, an Alkaline Phosphatase (AP) conjugated anti-DIG antibody (cat#11585762001) was incubated with the blots, and then followed with CDP-Star (supplied ready-to-use; cat#12041677001) for chemiluminescent detection of the DIG-labeled probe. Enzymatic reaction of CDP-Star reagent with alkaline phosphatase produces a light signal which can be detected by film or chemiluminescent-compatible instruments.
The Roche DIG system provides different kits and individual reagents for different applications. For Northern blot detection, in addition to the CDP-Star reagent, Roche also provides DIG Easy Hyb Granules (cat#11796895001) for hybridization. The Easy Hyb Granules only need to be reconstituted with DEPC water. DIG Easy Hyb saves all the troubles of making hybridization buffer and standardizes the hybridization process. Roche also provides a DIG Wash and Block Buffer Set (cat#11585762001), including10X blocking solution, wash buffer (for use after the antibody incubation) and detection buffer (used before application of CDP-Star). All of these buffers and reagents provide a standardized, efficient, fast and repeatable way to detect RNA expression in Northern blots.
Compared with the radioactive system for hybridization, Roche’s DIG System has the following advantages: First, because of the signal amplification achieved with the anti-DIG antibody, the system is very sensitive, even more sensitive than radioactivity. Therefore, it is good for detecting low copy DNA and low expression RNA. Second, compared with radioactive detection, the chemiluminescent signal used by the DIG system has short exposure times. Generally, radioactive labeling requires exposure times of hours or even days; however, for DIG system generally needs just several minutes. Third, it is safer and much more convenient to handle. Last, DIG probes can be made and stored in -80ºC for a long time, while radioactive probes generally need be made fresh each time because of decay. In addition, Roche provides very detailed and standardized protocols for each type of hybridization as well as troubleshooting instructions, which are very helpful.
For different experiments, the researcher needs to optimize the experimental conditions. For example, if you get high back-ground on a Northern blot by using DIG system, then you need to consider the following possibilities for trouble shooting:
Wrong type of nylon membrane: Some types of nylon membrane may cause high background. Use nylon membrane especially tested with the DIG-System from Roche Applied Science.
Inefficient hybridization: Recalculate hybridization temperature. Do not allow the membrane to dry between pre-hybridization and hybridization.
Ineffective stringency washes: Increase the stringency, such as increase temperature or lower the sodium concentration.
Prewarm wash solution to correct temperature.
Lower the probe concentration or the antibody concentration.