Riboprobe® in vitro Transcription Systems From Promega

Our lab performs in situ hybridization on a regular basis using RNA probes. We work with frozen sections of postmortem hippocampus from subjects with schizophrenia and bipolar disorder. After dipping the slides in photographic emulsion, we perform quantitative analyses of mRNA expression in GABA cells. In some cases we perform double in situ hybridization with a radioactive and non-radioactive probe. We have found that the Riboprobe® in vitro Transcription System from Promega consistently generates probes with high specific activity that are suitable for these experiments. The kits contain two RNA polymerases, either T7 and SP6 or T7 and T3, depending on the vector that you are using. Kits are also available with a single enzyme and additional enzymes can be purchased as needed. Therefore, you are not limited to vectors sold by Promega, but can use constructs in pBluescript or any other plasmid with dual opposable promoters. The kit contains everything required for the in vitro transcription except for the radioisotope. Contents include four unlabeled NTPs, one or two RNA polymerases, 5X transcription buffer, DTT, and RNasin RNase inhibitor. In addition, the kit includes RQ1 DNase which can be used to degrade the DNA template after the transcription reaction. We typically perform the reaction at 37ºC for two hours, adding an extra microliter of enzyme after one hour. After degrading the DNA template for 15 minutes, we purify the probe on a spin column (not part of the kit), and ethanol precipitate it. We use ³⁵S-UTP for our reactions but other isotopes and nucleotides can be used as well.

In terms of template preparation, there are a few options. Promega recommends phenol-chloroform extraction to purify the linearized template DNA. We have been using gel purification on a column. Former members of my lab had success using the linearized plasmid without any purification. Although linearized template is preferred in most cases, the transcription reaction can be carried out with circular plasmid DNA as well. The protocol recommends using 0.2 to 1 µg of template DNA in the reaction, but we have found that it is possible to use as little as 0.1 µg as long as sufficient probe is used for the hybridization. It is important to ensure that template DNA is completely linearized using a restriction enzyme that does not generate 3’ overhangs.

In addition to radioactive probes, we use the kit to synthesize digoxigenin labeled probes. In this case, we substituteRoche’s DIG RNA Labeling Mix for the NTPs included in the kit and perform the reaction as recommended. Likewise, it is possible to generate biotinylated probes with the kit.

In cases where larger quantities of RNA are required, Promega also makes a RiboMAX kit, which can generate milligrams of RNA. One disadvantage is that we use 20 µL of ³⁵S-UTP in a 10 µL reaction. Therefore, we have to dry the radioactive nucleotide in a vacuum centrifuge before setting up the reaction. This could present a problem for labs that do not have access to a vacuum centrifuge or other means of drying the isotope. It also adds extra time to the procedure.