Simultaneous Detection of Multiple mRNA Targets with Ribonuclease Protection Assays
|The ribonuclease protection assay (RPA) is an extremely sensitive method for detecting and quantitating RNAs (usually mRNAs) in a complex mixture of total cellular RNA (Melton et.al., 1984). The assay is at least 10 times as sensitive as standard Northern blotting, and it enables the investigator to use multiple probes in a single assay. In addition, the RPA is tolerant of partially degraded RNA, allows the use of sample sizes of up to 100 µg in order to further increase sensitivity, and can be used to perform mapping studies such as determining transcript initiation and termination sites and intron-exon boundaries. RPAs can also be used to discriminate between related mRNAs of similar size, which would migrate at similar positions on a Northern blot.|
In 1990, Ambion introduced the RPA™ Kit, the first commercially available ribonuclease protection assay kit. Having all the controls and optimized reagents available in kit form makes the technique more convenient and accessible to a wider range of researchers. Subsequently, Ambion improved this assay by developing a patented reagent, which simultaneously inactivates the RNase and precipitates the protected RNA fragments in a single step. This eliminates the Proteinase K/SDS digestion, phenol:chloroform extraction, and ethanol precipitation steps, and streamlines the procedure to a single tube assay. This technology is incorporated in Ambion's RPA II™ and RPA III™ ribonuclease protection assay Kits, which contain sufficient reagents to perform 100 assays, a positive control probe template and target mRNA, and comprehensive instruction manuals.
Tolerance for Partially Degraded Sample RNA
Simultaneous Detection of Multiple mRNA Targets
PharMingen has developed sets of ten to twelve probes templates for simultaneous use in RPAs. The probes are co-transcribed from the template set, column purified (no gel purification is used) and co-hybridized to an RNA sample. Figure 3 demonstates the successful use of these template sets in an RPA for simultaneous detection of multiple mRNA targets. In this example, an mApo-3 PharMingen template set was used in conjunction with Ambion's RPA III and MAXIscript™ transcription kits.
Lane 1: mApo-3 probe alone; Lane 2: mApo-3 probe + 10 µg CD4+h1 total RNA + RNase; lane 3: mApo-3 probe + 10 µg mouse fibroblast total RNA + RNase.
Using multiple probes clearly increases the quantity of data that can be gathered, but more importantly, increases the quality of the data that is obtained compared to analyzing mRNAs separately. Experimental variability is reduced or eliminated by measuring levels of multiple target mRNAs in the same sample, while one of the probes serves as an internal control by probing an RNA the level of which remains constant (e.g. glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-actin, or ribosomal RNA). Collectively, these advantages make RPAs the method of choice for detecting and quantitating multiple mRNAs.
- Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., and Green, M.R. (1984) Efficient In Vitro Synthesis of Biologically Active RNA and RNA Hybridization Probes From Plasmids Containing a Bacteriophage SP6 Promoter. Nuc. Acids Res. 12: 7035-7056.
- Ngai, J., Dowling, M.M., Buck, L., Axel, R., and Chess, A. (1993) The Family of Genes Encoding Odorant Receptors in the Channel Catfish. Cell 72: 657-666.