Abstract
Oligonucleotide probe tagging and reverse transcriptase polymerase-chain reaction (RT-PCR) are the most widely used techniques currently used for detecting and analyzing RNA. RNA detection using labeled oligonucleotide probe-based approaches is suitable for point-of-care (POC) applications but lacks assay sensitivity, whereas RT-PCR requires complex instrumentation. As an alternative, immunoassay detection formats coupled with isothermal RNA amplification techniques have been proposed for handheld assay development. In this chapter, we describe a robust technique comprising of: (a) target RNA tagging with a complementary oligonucleotide probe labeled with a hapten moiety to form a DNA/RNA duplex hybrid; (b) complexing the DNA/RNA duplex with a pre-coated antibody (Ab) directed at the hapten moiety; (c) sandwich complex formation with an Ab that selectively recognizes the DNA/RNA structural motif; and (d) detection of the sandwich complex using a secondary Ab enzyme conjugate targeting the anti-DNA/RNA Ab followed by standard enzyme-linked immunosorbent assay (ELISA) visualization.
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Acknowledgments
The authors acknowledge funding from the Johns Hopkins University Applied Physics Laboratory Independent Research and Development. We are very grateful to Dr. Stephen Leppla at National Institute of Allergy and Infectious Diseases (NIAID) for the generous gift of the S9.6 hybridoma cells for the anti-DNA-RNA Ab.
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Astatke, M., Tiburzi, O., Connolly, A., Robinson, M.L. (2024). RNA Analysis Using Immunoassay Detection Format. In: Astatke, M. (eds) RNA Amplification and Analysis. Methods in Molecular Biology, vol 2822. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3918-4_13
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DOI: https://doi.org/10.1007/978-1-0716-3918-4_13
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