Validation of miRNA Expression Profiling

In the stage 2 (Validation study), the expression of the selected miRNAs (hsa-miR-155-5p, MIMAT0000646; hsa-miR-181a, MIMAT0000256; and hsa-let-7a-5p, MIMAT 0000062) was measured by stem loop TaqMan RT-qPCR (Life Technologies, Carlsbad, CA, USA). Specific stem loop primers were used to synthetize the cDNA from 10 ng total RNA using the micro-RNA transcription kit (Life Technologies, CA, USA). RT-qPCR assays were carried out using predesigned assays (miR-155, ID: 002623; miR-181a, ID: 00048; and let-7a, ID: 000377) (Life Technologies CA, USA). RNU24 was selected as the most stable endogenous reference among 4 small nucleolar RNAs (RNU24, RNU6B, RNU58, RNU44), which were evaluated by using the GeNorm software [19 (link)]. All samples were assayed in duplicate, and the relative miRNA expression was analyzed using the comparative C_T method using the formula 2^−ΔCt [20 (link)].

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Cerda A., Amaral A.A., de Oliveira R., Moraes T.I., Braga A.A., Graciano-Saldarriaga M.E., Fajardo C.M., Hirata T.D., Bonezi V., Campos-Salazar A.B., Dorea E.L., Bernik M.M., Hirata M.H, & Hirata R.D. (2021). Peripheral Blood miRome Identified miR-155 as Potential Biomarker of MetS and Cardiometabolic Risk in Obese Patients. International Journal of Molecular Sciences, 22(3), 1468.

Publication 2021

TaqMan RT-qPCR micro-RNA transcription kit

Cdna Mirna Primers Small nucleolar rnas Stem Transcription

Corresponding Organization : Universidade de São Paulo

Other organizations : Hospital Universitário da Universidade de São Paulo

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression of the selected miRNAs (hsa-miR-155-5p, MIMAT0000646; hsa-miR-181a, MIMAT0000256; and hsa-let-7a-5p, MIMAT0000062)

control variables

RNU24 was selected as the most stable endogenous reference among 4 small nucleolar RNAs (RNU24, RNU6B, RNU58, RNU44), which were evaluated by using the GeNorm software

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

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