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Rapid and accurate quantification of isomiRs by RT-qPCR.

ABSTRACT: Currently, microRNAs (miRs) are annotated as a single defined sequence (canonical), even though high-throughput small RNA sequencing has identified miR isoforms (isomiRs) that differ from their canonical counterparts in length, sequence, or both. Here we describe a simple reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR)-based assay for quantification of the miR-100-5p_iso_3p:-2 variant. We chose miR-100-5p because the canonical sequence was underrepresented in our evaluation of human plasma. The quantification of miR-100-5p_iso_3 p:-2 from 57 plasma samples demonstrated high concordance between high-throughput RNA sequencing and RT-qPCR results (r = 0.55, p < 0.0001). Of note, we could not detect or quantify miR-100-5p in our plasma samples using a commercial TaqMan canonical miR-100-5p RT-qPCR kit. With these 57 samples, we also adapted this assay to specifically quantify the canonical sequences of miR-122-5p and miR-192-5p. Similar to the results obtained with miR-100-5p_iso_3p:-2, RT-qPCR results for miR-122-5p and miR-192-5p highly correlated with high-throughput RNA sequencing data (miR-122-5p: r = 0.44, p = 0.0005; miR-192-5p: r = 0.72, p < 0.0001). The assay described here can be easily adapted to many different identified isomiRs. Because of the high specificity of isomiRs, their reliable RT-qPCR-based quantification could provide greater resolution and higher accuracy than using canonical sequences.

SUBMITTER: Franco S

PROVIDER: S-EPMC9568571 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Rapid and accurate quantification of isomiRs by RT-qPCR.

Franco Sandra S Pluvinet Raquel R Sanchez-Herrero Jose Francisco JF Sumoy Lauro L Martinez Miguel Angel MA

Scientific reports 20221014 1

Currently, microRNAs (miRs) are annotated as a single defined sequence (canonical), even though high-throughput small RNA sequencing has identified miR isoforms (isomiRs) that differ from their canonical counterparts in length, sequence, or both. Here we describe a simple reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR)-based assay for quantification of the miR-100-5p_iso_3p:-2 variant. We chose miR-100-5p because the canonical sequence was underrepresented in our evaluatio ...[more]

PMID: 36241713

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Project description:ObjectivesWe explore the importance of SARS-CoV-2 sentinel surveillance testing in primary care during a regional COVID-19 outbreak in Austria.DesignProspective cohort study.SettingA single sentinel practice serving 22 829 people in the ski-resort of Schladming-Dachstein.ParticipantsAll 73 patients presenting with mild-to-moderate flu-like symptoms between 24 February and 03 April, 2020.InterventionNasopharyngeal sampling to detect SARS-CoV-2 using real-time reverse transcriptase-quantitative PCR (RT-qPCR).Outcome measuresWe compared RT-qPCR at presentation with confirmed antibody status. We split the outbreak in two parts, by halving the period from the first to the last case, to characterise three cohorts of patients with confirmed infection: early acute (RT-qPCR reactive) in the first half; and late acute (reactive) and late convalescent (non-reactive) in the second half. For each cohort, we report the number of cases detected, the accuracy of RT-qPCR, the duration and variety of symptoms, and the number of viral clades present.ResultsTwenty-two patients were diagnosed with COVID-19 (eight early acute, seven late acute and seven late convalescent), 44 patients tested SARS-CoV-2 negative and 7 were excluded. The sensitivity of RT-qPCR was 100% among all acute cases, dropping to 68.1% when including convalescent. Test specificity was 100%. Mean duration of symptoms for each group were 2 days (range 1-4) among early acute, 4.4 days (1-7) among late acute and 8 days (2-12) among late convalescent. Confirmed infection was associated with loss of taste. Acute infection was associated with loss of taste, nausea/vomiting, breathlessness, sore throat and myalgia; but not anosmia, fever or cough. Transmission clusters of three viral clades (G, GR and L) were identified.ConclusionsRT-qPCR testing in primary care can rapidly and accurately detect SARS-CoV-2 among people with flu-like illness in a heterogeneous viral outbreak. Targeted testing in primary care can support national sentinel surveillance of COVID-19.

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Rapid and accurate quantification of isomiRs by RT-qPCR.

Publications

Rapid and accurate quantification of isomiRs by RT-qPCR.

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