Rapid and accurate quantification of isomiRs by RT-qPCR
Franco Cirera, Sandra (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Pluvinet, Raquel (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Sanchez-Herrero, Jose Francisco (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Sumoy, Lauro (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Martinez, Miguel Angel (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)

Fecha:	2022
Resumen:	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.
Ayudas:	Agencia Estatal de Investigación PID2019-103955RB-I00
Derechos:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Lengua:	Anglès
Documento:	Article ; recerca ; Versió publicada
Materia:	Biological techniques ; Microbiology ; Molecular biology
Publicado en:	Scientific reports, Vol. 12 (october 2022) , ISSN 2045-2322

DOI: 10.1038/s41598-022-22298-7
PMID: 36241713

8 p, 1.6 MB

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Documentos de investigación > Documentos de los grupos de investigación de la UAB > Centros y grupos de investigación (producción científica) > Ciencias de la salud y biociencias > Institut d'Investigació en Ciencies de la Salut Germans Trias i Pujol (IGTP)
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