The DIG RNA Labeling Kit SP6/T7 From Roche

The DIG RNA Labeling Kit is based on the in vitro transcription of a template cDNA. cDNA is cloned into the polylinker site of a transcription vector (supplied) containing SP6 and T7 promoters. Digoxigenin (DIG) -labeled UTP is incorporated (approximately in a 1:2 ratio with UTP) into the transcript, generating DIG-labeled RNAs. The kit provides everything to produce enough probe to use in different applications, such as probing Northern blots, RNase protection assays, and in situ hybridization. The kit contains 2 controls (labeled and unlabeled complementary RNAs) to test labeling efficiency using positively-charged nylon membrane (not supplied).The following equipment is required: a temperature-controlled water bath to perform the labeling reaction and an UV-transilluminator to crosslink RNA to the membrane. Once the linearized vector, containing the template of interest, has been purified, the procedure is very simple. All steps, however, need to be carried out on ice. Two different RNase-free tubes are required: one for the sense probe and another for the antisense probe. The first step of the protocol is to add enough linearized template to have 1 ug of it in 13 ul of diethylpyrocarbonate (DEPC) treated water. Then, in the sterile, RNase-free tubes, add 2 ul of the dNTP labeling mixture, 2 ul of transcription buffer, 1 ul of RNase inhibitor, 2 ul of the appropriate SP6 or T7 RNA polymerase and then incubate for 2 h at 37ºC. To stop the reaction, add 2 ul of 0.2 M EDTA. The reaction may be scaled up to increase the amount of labeled RNA, but we don’t suggest it if different genes have to be investigated, to not increase the cost/reaction ratio.

To determine the yield of labeled RNA, the direct detection method is performed. A series of dilutions (suggested in the datasheet) of probes and control labeled RNA are spotted on nylon membrane, then crosslinked with an UV transilluminator and detected using an anti-DIG alkaline phosphatase conjugated antibody (not supplied). We generally perform the last step using NBT/BCIP (not supplied in the kit) as substrate. This method is less expensive than the chemiluminescent reaction with CSPD suggested by the datasheet, although quite less sensitive. The labeled sense and antisense RNA probes are then ready for downstream applications (i.e. northern blot, in situ hybridization, etc.).

We used the DIG RNA labeling kit to produce several probes to study the peptide-receptor system through non-radioactive in situ hybridization technique (ISH): We constructed 300 base pair probes to reveal the distribution of 2 closely related neuropeptides, the pituitary adenylate cyclase-activating polypeptide (PACAP) and the vasoactive intestinal polypeptide (VIP), in the brain of vertebrates. We also performed ISH and Northern blot hybridization to detect the distribution of their receptors (VPAC₁, VPAC₂ and PAC₁). Moreover, we used this kit to detect different nicotinic acetylcholine receptor subunits in several tissues of amphibians (1). We always obtained good yields of probes (3-4 ug of probe per 1 ug of purified template), though less then the standard quantity stated in the datasheet (10ug of probe per 1 ug of purified template); this was probably due to the non-standard conditions used in our experiments (e.g. different plasmid used, different kind of template purification). To detect the DIG RNA probes with both ISH and Northern techniques, we employed immunological detection with an anti-DIG antibody conjugated with alkaline phosphatase (Roche) and BM purple (Roche) or NBT/BCIP revelation.

To conclude, this kit provides is a good tool when performing extensive studies of messenger expression, since it gives enough probe to carry out several tests (for ISH the standard 20 ul reaction is sufficient for 10-15 experiments). In contrast, a bad feature of the kit is that many of the reagents necessary for determining labeling efficiency and final probe concentration are not provided.

Salvatore Valiante, PhD
University of Naples “Federico II”
Department of Biological Sciences
Section of Evolutionary and Comparative Biology

¹Salvatore Valiante, Anna Capaldo, Francesca Virgilio, Rosaria Sciarrillo, Maria De Falco, Flaminia Gay, Vincenza Laforgia, Lorenzo Varano. “Distribution of alpha-7 and alpha-4 nicotinic acetylcholine receptor subunits in several tissues of Triturus carnifex (Amphibia, Urodela),” Tissue and Cell 36:391–398, 2004.