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Nucleic Acids Res. 2014 Aug;42(14):9146-57. doi: 10.1093/nar/gku636. Epub 2014 Jul 25.

Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay.

Author information

1
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA California Institute for Biomedical Research (Calibr), La Jolla, CA 92307, USA.
2
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA The Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, CA 92037, USA.
3
California Institute for Biomedical Research (Calibr), La Jolla, CA 92307, USA.
4
The Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, CA 92037, USA.
5
The Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, CA 92037, USA Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA schultz@scripps.edu.
6
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA California Institute for Biomedical Research (Calibr), La Jolla, CA 92307, USA schultz@scripps.edu.

Abstract

A sensitive and highly multiplex method to directly measure RNA sequence abundance without requiring reverse transcription would be of value for a number of biomedical applications, including high throughput small molecule screening, pathogen transcript detection and quantification of short/degraded RNAs.

R NA A:

nnealing, S: election and L: igation (RASL) assays, which are based on RNA template-dependent oligonucleotide probe ligation, have been developed to meet this need, but technical limitations have impeded their adoption. Whereas DNA ligase-based RASL assays suffer from extremely low and sequence-dependent ligation efficiencies that compromise assay robustness, Rnl2 can join a fully DNA donor probe to a 3'-diribonucleotide-terminated acceptor probe with high efficiency on an RNA template strand. Rnl2-based RASL exhibits sub-femtomolar transcript detection sensitivity, and permits the rational tuning of probe signals for optimal analysis by massively parallel DNA sequencing (RASL-seq). A streamlined Rnl2-based RASL-seq protocol was assessed in a small molecule screen using 77 probe sets designed to monitor complex human B cell phenotypes during antibody class switch recombination. Our data demonstrate the robustness, cost-efficiency and broad applicability of Rnl2-based RASL assays.

PMID:
25063296
PMCID:
PMC4132746
DOI:
10.1093/nar/gku636
[Indexed for MEDLINE]
Free PMC Article

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