United States [ Change ]Invader® miRNA
Third Wave has developed reagents* based on our Invader® chemistry for the detection and quantitation of a range of microRNAs, or miRNAs. This emerging area of nucleic acid testing is important and valuable. Changes in miRNA levels are associated with the development of cancer and other diseases.1-2
MicroRNA Overview and Background
Many hundreds of miRNAs have been identified in plants, animals, and several viruses, and a variety of functions have been shown to be influenced by them, including the regulation of cellular differentiation, proliferation and apoptosis.2 Because of the roles of miRNAs in controlling gene expression, it is not surprising that alterations in the amounts of particular miRNAs are associated with certain cancers,3-10 and several laboratories have suggested using the expression levels of these miRNAs as prognostic indicators of disease.
The small size of miRNAs can make it difficult to quantify their levels, particularly when very little sample is available for analysis. Some assays resort to using very large samples whereas others rely on amplification of the sequence by a series of (often non-quantitative) steps such as ligation followed by the polymerase chain reaction. While the global parallel analysis of many miRNAs can be useful for many applications, often the investigator wishes to compare the amounts of one miRNA, or of a representative subset of miRNAs, isolated from a variety of samples or under a variety of conditions. In these latter cases it is useful to have a reliable, quantitative assay that is adaptable to repeated analysis of a few chosen sequences.
Third Wave has developed an assay for sensitive and specific detection and quantification of miRNAs. This second-generation Invader® miRNA assay is based upon the Invader Plus® chemistry11 and has the ability to detect and reproducibly quantify as few as 50-100 molecules of an individual miRNA. It distinguishes between miRNAs and their precursors, as well as between closely related miRNA isotypes. The assay is rapid and can be performed in detergent lysates of cells without need for total RNA isolation. The Invader® miRNA assay is configured in a single-addition, single-tube format eliminating the transfer of reverse transcription product into later steps. Furthermore, it does not require individual labeled probes or expensive instrumentation thus making it a good choice for low-cost high-throughput detection and quantitation of miRNAs. Its data can be acquired and analyzed using either end-point or real-time fluorescence detection and is compatible with most commercially available plate readers and real-time fluorescence instruments. The Invader® miRNA assay can also be performed in a single-tube biplex format allowing for simultaneous detection of miRNAs of interest and normalizing the data to an endogenous RNA control.
Schematic of the Invader® microRNA Reaction:
1. Bartel, D.P. (2004) Cell. 116, 281-97.
2. Ambros, V. (2004) Nature. 431, 350-5.
3. Calin, G.A., Dumitru, C.D., Shimizu, M., Bichi, R., Zupo, S., et al. (2002) Proc Natl Acad Sci U S A. 99, 15524-9.
4. Michael, M.Z., O'Connor, S.M., van Holst Pellekaan, N.G., Young, G.P., et al. (2003) Mol Cancer Res. 1, 882-91.
5. Takamizawa, J., Konishi, H., Yanagisawa, K., Tomida, S., Osada, H., et al. (2004) Cancer Res. 64, 3753-6.
6. Johnson, S.M., Grosshans, H., Shingara, J., Byrom, M., Jarvis, R., et al. (2005) Cell. 120, 635-47.
7. Eis, P.S., Tam, W., Sun, L., Chadburn, A., Li, Z., et al. (2005) Proc Natl Acad Sci U S A. 102, 3627-32.
8. He, L., Thomson, J.M., Hemann, M.T., Hernando-Monge, E., Mu, D., et al. (2005) Nature. 435, 828-33.
9. Metzler, M., Wilda, M., Busch, K., Viehmann, S. and Borkhardt, A. (2004) Genes Chromosomes Cancer. 39, 167-9.
10. O'Donnell, K.A., Wentzel, E.A., Zeller, K.I., Dang, C.V. and Mendell, J.T. (2005) Nature. 435, 839-43.
11. Allawi, H.T., Li, H., Sander, T., Aslanukov, A., Lyamichev, V.I., et al. (2006) J. Clin. Microbiol. 44, 3443-3447.
The Invader® chemistry is protected by foreign and domestic patents.
*For Research Use Only. Not for Use in Diagnostic Procedures.
Invader® and Cleavase® are registered trademarks of Third Wave Technologies, Inc.
Third Wave's Invader® miRNA assay is covered by pending domestic and foreign patent applications, including WO 04/057017 and US 20050074788.