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

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


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • High-Throughput Nucleotide Sequencing
  • Humans
  • Immunoglobulin Class Switching
  • Molecular Probe Techniques
  • RNA Ligase (ATP)
  • Sensitivity and Specificity
  • Sequence Analysis, RNA / methods*
  • Viral Proteins


  • Viral Proteins
  • RNA Ligase (ATP)
  • bacteriophage T4 RNA ligase 2