Dataset Information

A Fluorescent Reverse-Transcription Assay to Detect Chemical Adducts on RNA.

ABSTRACT: Herein, we detail a novel reverse-transcription (RT) assay to directly detect chemical adducts on RNA. We optimize a fluorescence quenching assay to detect RT polymerization and employ our approach to detect N¹-alkylation of inosine, an important post-transcriptional modification, using a phenylacrylamide as a model compound. We anticipate our approach can be expanded to identify novel reagents that form adducts with RNA and further explored to understand the relationship between RT processivity and natural post-transcriptional modifications in RNA.

SUBMITTER: Falco N

PROVIDER: S-EPMC10010264 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A Fluorescent Reverse-Transcription Assay to Detect Chemical Adducts on RNA.

Falco Natalie N Garfio Chely M CM Spitalny Leslie L Spitale Robert C RC

Biochemistry 20220725 16

Herein, we detail a novel reverse-transcription (RT) assay to directly detect chemical adducts on RNA. We optimize a fluorescence quenching assay to detect RT polymerization and employ our approach to detect N<sup>1</sup>-alkylation of inosine, an important post-transcriptional modification, using a phenylacrylamide as a model compound. We anticipate our approach can be expanded to identify novel reagents that form adducts with RNA and further explored to understand the relationship between RT p ...[more]

PMID: 35876726

Similar Datasets

Project description:BackgroundTick-borne encephalitis virus (TBEV) is a causative agent of acute central nervous system disease in humans. It has three subtypes, far eastern (FE), Siberian (Sib) and European (Eu) subtypes, which are distributed over a wide area of Europe and Asia. The objective of this study was to develop a simple and rapid assay for the detection of TBEV RNA by using reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) method that can differentiate the three subtypes of TBEV and can be used for clinical diagnosis and epidemiological study.MethodsPrimers for TBEV-specific and subtype-specific RT-LAMP assay were designed to target the consensus sequence in NS1 of all subtypes and the consensus sequence in the E gene of each subtype, respectiveluy. In vitro transcribed RNA of Oshima strain that belongs to FE subtype was serially diluted and used to examine the sensitivity of the assay. Cross-reactivity of subtype-specific RT-LAMP assay was tested by using the RNA of Oshima and Sofjin (FE), IR-99 (Sib) and Hochosterwitz (Eu) strains. RNA extracted from the mixtures of TBEV and ticks, and of TBEV and human blood, and the mouse tissues infected with TBEV, were evaluated in the assay. Positive amplification was observed by real-time monitoring of turbidity and by visual detection of color change.ResultsThe sensitivity of TBEV-specific RT-LAMP assay was 102 copies of target RNA per reaction volume. FE-specific RT-LAMP assay amplified viral genes of Oshima and Sofjin strains but not of IR-99 and Hochosterwitz strains, and of Japanese encephalitis virus. RT-LAMP assay for Sib and for Eu specifically amplified viral genes of IR-99 and Hochosterwitz strains, respectively. We also showed that tick or human blood extract did not inhibit the amplification of viral gene during the assay. Furthermore, we confirmed that the TBEV RT-LAMP could detect virus RNA from peripheral and central nervous system tissues of laboratory mice infected with TBEV.ConclusionTBEV RT-LAMP assay offers a sensitive, specific, rapid and easy-to-handle method for the detection of TBEV RNA in tick samples and this may be applied in the clinical samples collected from TBE-suspected patients.

Dataset Information

A Fluorescent Reverse-Transcription Assay to Detect Chemical Adducts on RNA.

Publications

A Fluorescent Reverse-Transcription Assay to Detect Chemical Adducts on RNA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets