RNA-mediated TILDA for improved cell capacity and enhanced detection of multiply-spliced HIV RNA

Hannah M. Pezzi, Scott M. Berry, David J. Beebe, Rob Striker

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Quantification of the HIV viral reservoir is critical to understanding HIV latency, advancing patient care and ultimately achieving a cure. To quantify the reservoir, a new metric was recently introduced, which quantified cells carrying multiply spliced HIV RNA. The developed assay, Tat/rev Induced Limiting Dilution Assay (TILDA), enables quantification of cells containing multiply-spliced HIV RNA events as an indicator of reservoir size. Due to TILDA's reliance on a limiting dilution format paired with the rarity of target events, numerous individual reactions are required to obtain a single endpoint. The current assay embodiment uses a whole cell input to detect target RNA sequences without the traditional preceding nucleic acid purification steps. Thus, while the direct measurement of target events from whole cells significantly streamlines the workflow, there is a cost in sensitivity and assay throughput. Here, we apply a new technique for rapid RNA isolation, Exclusion-Based Sample Preparation, to TILDA, with the goal of alleviating these limitations without significantly adding to the workflow. By combining TILDA with multiplexed RNA extraction enabled by exclusion-based sample preparation, assay sensitivity and capacity are improved while maintaining assay simplicity, advancements that could facilitate eventual clinical implementation in detecting rare events in patients.

Original languageEnglish
Pages (from-to)876-884
Number of pages9
JournalIntegrative Biology (United Kingdom)
Volume9
Issue number11
DOIs
StatePublished - Nov 2017

Bibliographical note

Funding Information:
The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 LAV infected Jurkat E6 Cells (J1.1) from Dr Thomas Folks.24 The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: ACH-2 from Dr Thomas Folks.25,26 This work was supported by the University of Wisconsin Foundation, Wisconsin Partnership Collaborative Health Sciences Program (HP, SB, DB, RS), NIH R01CA181648-Lang/Berry CTC R01 (HP, SB), and the University of Wisconsin Carbone Cancer Center Cancer Center Support Grant P30 CA014520 (DB). Additionally, the authors would like to thank all study participants for their contribution in making this study possible. The authors would also like to thank Wynne Moss and Nathan Teachout for assistance in sample processing as well as David John Guckenberger for contribution and assistance in developing the automated RNA extraction platform.

Publisher Copyright:
© 2017 The Royal Society of Chemistry.

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry

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