DIG RNA Labeling Mix From Roche

We have routinely used the Roche DIG-UTP RNA Labeling Mix to synthesize single stranded, DIG-labeled RNA probes for in-situ hybridization (ISH) in zebrafish embryos. Digoxigenin (DIG) is a small organic molecule, bound to UTP in this kit, which is incorporated into transcribed RNA by RNA polymerases. The DIG can then be detected through the use of a specific anti-DIG antibody and standard immunohistochemical techniques. In addition to in vivo ISH, the DIG RNA Labeling Kit could be used to generate single-stranded probes for techniques such as Northern blots (where subsequent DIG visualization can be used to identify specific products), chromosome or DNA staining, or bacterial colony lifts.

The premise of the kit is quite simple. Complementary, single-stranded RNA probes are synthesized from a defined DNA template, usually a linearized plasmid, by using T7, T3 or Sp6 RNA polymerases. RNA transcribed from this vector will include the DIG label, which we have found to be very easily detectable in ISH experiments by using an anti-DIG alkaline-phosphatase conjugated antibody. We visualize through a BCIP (5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt) and NBT (Nitro-Blue Tetrazolium Chloride) colorimetric reaction.

Technically, the kit is very easy to use, although it is extremely important to take great care in the preparation of the RNA probes, as contamination must be avoided at all costs. This begins with the preparation of the template DNA, which should be completely linearized, very pure, and the exact concentration must be known. It is important to check this before the transcription reaction is set up. Roche recommend that no more than 1 ug of template be used for transcription, as any more can inhibit probe synthesis. The reaction itself is quite straightforward: DNA template is mixed with nucleotides, DIG, buffer and RNA polymerase, and incubated at 37ºC. Although the recommended time is 2 hours, we find that incubation for 2.5 hours dramatically increases the yield of probe, particularly when using Sp6 polymerase. DNA template is removed by DNAse treatment (important to not incubate this step of the reaction for too long, as the RNA probe can also be digested), and finally, the RNA is precipitated in order to remove unincorporated nucleotides from the reaction. Although several methods exist for this procedure, including purification through Sephadex columns, we find that overnight precipitation with 4 M LiCl and 100% Ethanol at -20 ºC gives the best results. Again, the concentration of LiCl is crucial for successful precipitation; DIG immunoreactivity is not compromised by this precipitation.

In summary, the DIG RNA Labeling Mix is a very neat system to generate labeled RNA probes. Under optimized conditions, we find the system to be very reliable and efficient; although in my experience, this is the molecular biology technique that requires the greatest precision in terms of volumes, concentrations and incubation times. Using these probes, all my ISH experiments have worked very well, and although all the components of the probe synthesis and subsequent visualization are not cheap, the results which can be generated are very impressive and reproducible.