title: Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay.
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.
  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.
authors: Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA California Institute for Biomedical Research (Calibr), La Jolla, CA 92307, USA.
authors: H Benjamin
authors: HB
authors: Larman
authors: 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.
authors: Erick R
authors: ER
authors: Scott
authors: California Institute for Biomedical Research (Calibr), La Jolla, CA 92307, USA.
authors: Megan
authors: M
authors: Wogan
authors: The Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors: Glenn
authors: G
authors: Oliveira
authors: 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.
authors: Ali
authors: A
authors: Torkamani
authors: 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.
authors: Peter G
authors: PG
authors: Schultz
mesh_terms: D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D017578:Immunoglobulin Class Switching; D015336:Molecular Probe Techniques; D011130:RNA Ligase (ATP); D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000379:methods*; D014764:Viral Proteins
chemical_list: D014764:Viral Proteins; D011130:RNA Ligase (ATP); C467724:bacteriophage T4 RNA ligase 2