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qInvader™ Technology*

Third Wave's new qInvader™ reagents* detect and quantify nucleic acids such as microRNAs (miRNAs), messenger RNAs (mRNA), and DNA over a very large range of concentrations. Changes in the amounts of specific nucleic acids are associated with the development of cancer and other diseases1-2 so qInvader's real-time fluorescent read-out can be a very useful tool in understanding these conditions.

MicroRNAs

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.

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While the global parallel analysis of many miRNAs can be useful for many applications, often the investigator wishes to compare the amounts of only 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.

Messenger RNAs

The same protocols used to assay miRNAs by qInvader™ can also be used in analysis of messenger RNAs. For example, the ease, sensitivity and reliability of the assay allows for the parallel analysis of many samples isolated under a variety of conditions or during a time course. Also, the high specificity of the assay allows quantification of particular isoforms of alternatively spliced or edited mRNAs.

Third Wave's qInvader™ miRNA/mRNA assays were developed to be sensitive and specific for the detection and quantification of miRNAs and mRNAs. Based upon the Invader Plus® chemistry11, it has the ability to detect and reproducibly quantify as few as 10 molecules of an individual RNA species. It distinguishes between miRNAs and their precursors, as well as between closely related miRNA or mRNA isotypes. The qInvader™ assay can be performed in a single-tube biplex format, which allows for simultaneous detection of two target nucleic acids of interest. That facilitates direct comparison of relative gene expression levels as well as normalization for variations in sample preparation. The qInvader™ assay is rapid and can be performed in detergent lysates of cells without need for total RNA isolation, and it does not use individual labeled probes. Results can be acquired and analyzed using real-time fluorescence detection instruments and the system is compatible with most commercially available real-time fluorescence instruments.

DNA

Third Wave's qInvader™ Technology can be used for many DNA applications such as qualitative DNA detection or quantitative copy number determination, genotyping and single-nucleotide polymorphism detection. The qInvader™ reagents combines the power of PCR amplification with the structure-specific detection and signal generation and amplification of Invader® technology. qInvader™ Technology offers high sensitivity and specificity, low background, large quantitative dynamic range.

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.

Invader® and Invader Plus® chemistries are protected by foreign and domestic patents.

*For Research Use Only. Not for Use in Diagnostic Procedures.

qInvaderTM is a trademark for Third Wave Technologies

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.