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

H Benjamin Larman1, Erick R Scott2, Megan Wogan3

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A new RNA ligase 2 (Rnl2)-based method enables direct RNA measurement without reverse transcription. This robust and cost-efficient RNA-templated ligation sequencing (RASL-seq) assay offers high sensitivity for biomedical applications.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Direct RNA sequence abundance measurement is valuable for applications like high-throughput screening and pathogen detection.
  • Existing RNA template-dependent oligonucleotide probe ligation (RASL) assays have technical limitations, including low and sequence-dependent ligation efficiencies.

Purpose of the Study:

  • To develop a sensitive and robust method for direct RNA quantification without reverse transcription.
  • To overcome the limitations of previous RASL assays using RNA ligase 2 (Rnl2).

Main Methods:

  • Development of Rnl2-based RASL assays for direct RNA ligation to oligonucleotide probes on an RNA template.
  • Optimization of probes for analysis by massively parallel DNA sequencing (RASL-seq).
  • Assessment of a streamlined Rnl2-based RASL-seq protocol in a small molecule screen monitoring human B cell phenotypes.

Main Results:

  • Rnl2-based RASL demonstrated sub-femtomolar transcript detection sensitivity.
  • The method allows rational tuning of probe signals for RASL-seq.
  • The streamlined protocol proved robust, cost-efficient, and broadly applicable in a complex biological screen.

Conclusions:

  • Rnl2-based RASL-seq is a sensitive, robust, and cost-effective method for direct RNA quantification.
  • This assay overcomes previous technical limitations and has broad applicability in biomedical research.
  • The method is suitable for high-throughput screening and detection of various RNA types.