The ribonuclease protection assay (RPA) is a highly sensitive and specific method for detecting and quantifying mRNA species in a mixture of total RNA or polyA RNA.
For the RPA, a radioactive- or biotin-labeled RNA probe is synthesized from a DNA template that is complementary to the target mRNA under investigation. Multi-probe detection is possible through the generation of a series of templates with different lengths, each representing a distinct mRNA species. For example, we and others used this method successfully to examine cytokine and chemokine gene expression repertoires in a variety of experimental model systems with probes from BD Biosciences, as well as with self-generated probes.
The labeled probe and sample RNA are incubated under conditions which favor hybridization of complementary RNA sequences. After digestion with RNase to remove unhybridized RNA, the hybridized sample is separated on a polyacrylamide gel and the ‘protected’ RNA is detected either directly by autoradiography or after transfer to a membrane by probing with streptavidin-HRP and chemiluminescent (e.g. ECL) reagents. The identity and quantity of each mRNA species in the original RNA sample can then be determined based on the signal intensities given by the appropriately sized, protected probe fragment bands. Template for the analysis of L32 and GADPH house-keeping genes are included to allow assessment of total RNA levels for normalizing sampling and technical errors.
The most sensitive kit available is the RPAIII™ Kit, which includes all reagents necessary to perform RPA, but requires a labeled nucleotide antisense mRNA probe. It includes improved hybridization and digestion buffers that provide twice the sensitivity of the RPA II™; the RPAIII™ Kit can be used to detect down to 4,000 copies of mRNA. The RPA III™ Kit is available in 2 sizes to perform either 35 or 100 reactions. When using BD Biosciences’ Multi-Probe Template Set, a slightly different protocol than the one provided in the kit should be followed; the adapted protocol is available on Ambion’s website.
I used the RPAIII™ kit with a radiolabeled mck-3b mouse cytokine multiprobe template from BD Biosciences to detect LTá, LTâ, TNF, IL-6, IFNã, IFNâ, TGFâ1, TGFâ2, TGFâ3, MIF, L32 and GAPDH in total RNA from spinal cord and brain tissue of mice with either experimental autoimmune encephalomyelitis, an inflammatory demyelinating autoimmune disease, or healthy control mice. For the assay, I isolated total RNA from the tissue and analyzed the RNA for purity and degradation on a formamide gel. Care has to be taken during the whole procedure when working with RNA to avoid contamination and degradation with RNAse: wear gloves, use sterile plastic-ware and RNAse-free reagents. Initially, I used the protocol provided in the kit, where the total RNA is co-precipitated with the labeled probe and hybridized at 42°C. When my labeled probe did not hybridize with the tissue RNA, I bought new reagents and tried different RNA concentrations. Finally, when I called, technical service at Ambion pointed out that they have a separate protocol optimized for using the Multi-Probe Set from BD Biosciences (no precipitation, 56°C hybridization). They also helped me to appropriately adjust the conditions for my samples; I adjusted the amount of labeled probe and sample, as well as gel running/loading conditions in order to visualize a useful ‘ladder’ on the film. For the different RNA samples, I adjusted the concentrations in several experiments but finally succeeded when using between 10 and 20 µg of RNA with 2 µl of the labeled probe (3x105 cpm) for each reaction. One important step is also not to over-dry the pellet before re-suspending it in loading buffer. If overdried, it can be difficult to completely be re-suspended the pellet; thus it can remain in the gel wells, lowering the amount of detected signal in the gel.
Even though it may be time-consuming to establish the right concentrations of total RNA and labeled probe in the beginning, the steps of the protocol are easy to follow and well-established to provide consistent and reliable results in detecting multiple mRNA species in the same sample RNA. This not only limits the amount of sample needed to detect several mRNA species, but also the radioactivity and time to perform the experiments. I highly recommend this kit (also for less experienced researchers in molecular biology), since it is an easy to use and fast procedure, which also allows you to process several samples at the same time. The kit includes a good protocol and troubleshooting guide; in addition Ambion’s technical service is very helpful.