During my work, I had to make RNA probes for in situ hybridization in
Xenopus embryos and I used this kit. This kit allows the DNA template to be transcribed and cloned into the polylinker site of appropriate transcription vectors, which contain promoters for SP6 and/or T7 RNA polymerases. After cloning and linearization at a suitable site, RNA is then transcribed in the presence of DIG-UTP. Under standard conditions, approximately 10 µg of full-length DIG-labeled RNA may be transcribed from 1 µg template.
This kit contains two types of cloning vectors pSPT18 DNA, (40 µl, 0.25 mg/ml) and pSPT19 DNA (40 µl, 0.25 mg/ml). In order to produce RNA probes, your DNA must be cloned in one of these vectors. Also, the kit includes two types of DNA controls. Control DNA 1 is pSPT18-Neo, (20 µl, 0.25 mg/ml) cleaved with PvuII for trancsription by T7 RNA polymerase. Transcription of Control DNA I with T7 RNA polymerase produces DIG-labeled “antisense” Neo transcripts 760 bp in length. Control DNA 2 is pSPT19-Neo (20 µl, 0.25 mg/ml) cleaved with PvuII with SP6. Transcription of Control DNA 2 with SP6 RNA polymerase makes DIG-labeled “antisense” Neo transcripts 760 bp in length. Cleavage with PvuII of both controls produces two fragments. In the case of control DNA 1, the resulted fragments are 798 bp and 3281 bp. Cleavage of conrol DNA 2 makes 316 bp and 3761 bp fragments. Other controls include DIG-labeled control RNA (100 µl, 100 ng/µl), which is DIG-labeled “antisense” neo RNA, and an unlabeled control RNA (20 µl, 200 µg/ml), which is neo poly (A) “sense” RNA. Both RNA controls are supplied in dimethyldicarbonate-treated double-distilled water. Other componets of this kit include: NTP Labeling Mixture, 40 µl, 10x conc. (10 mM ATP, CTP, GTP, 6.5 mM UTP, 3.5 mM DIG-11-UTP); pH 7.5 Transcription Buffer 40 µl, (10x conc); DNase I, RNase-free (40 µl, 10 U/µl); RNase Inhibitor, (20 µl, 20 U/µl); SP6 RNA Polymerase ( 20 µl, 20 U/µl); T7 RNA Polymerase, (20 µl, 20 U/µl).
In order to DIG-label your RNA, you have to clone your chosen insert into one of the included vectors, then linearize it with one of the enzymes from the polycloning site and clean the resulted DNA. I usually used High Pure PCR Product Purification Kit (Roche Applied Science) which has cut-off of 17 bp for purification of the linearized fragments. In order to avoid trancsription of undesirable sequences, it better to use restriction enzyme that produce 5’ overhangs. According to the manual, each reaction uses about 1 µg of linearized DNA, but I found that DNA from 0.5 up to 2 µg gave satisfactory results.
About 1 µg of linearized DNA should be dissolved into 13 µl of double distil water. I also found that heating the DNA (5 min, 70°C) gave better results, probably due to the DNA strands annealing. I then placed the test tube containing the heated DNA on ice and added the other components of the kit according the manual. After spinning down, the reaction is then incubated about 2 h at 37°C. Actually, I didn’t see significant increase in the amount of trancscribed product after 1 hour of the incubation. However, addition of 0.5µl of the enzyme after 1 hour of incubation usually increased the amount of the resulting RNA. Because the resulting DIG-labeled RNA was made to use as probe in in situ hybridization, the RNA was treated with DNase I (15 min at 37°C) and then purified by phenol-chloroform extraction and subsequent ethanol precipitation.The purified DIG-labeled RNA was resuspended in double-distilled sterile water:20XSSC:formaldehyde (5:3:2), aliqouted and stored at -80°C.
In conclusion, the DIG RNA Labeling Kit is a convenient, reliable, easy to use product and gives excellent reproducibility. I highly recommend it.
Neuroimmunology Laboratory Manager
Department of Neurology
Sourasky Tel-Aviv Medical Research Center