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Assay reproducibility in clinical studies of plasma miRNA

Rice, Jonathan ; Roberts, Henry ; Burton, James ; Pan, Jianmin ; States, Vanessa ; Rai, Shesh N ; Galandiuk, Susan Hoheisel, Jörg D.

PloS one, 2015-04, Vol.10 (4), p.e0121948-e0121948 [Periódico revisado por pares]

United States: Public Library of Science

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Título:
Assay reproducibility in clinical studies of plasma miRNA
Autor: Rice, Jonathan ; Roberts, Henry ; Burton, James ; Pan, Jianmin ; States, Vanessa ; Rai, Shesh N ; Galandiuk, Susan
Hoheisel, Jörg D.
Assuntos: Aged ; Aged, 80 and over ; Assaying ; Bioinformatics ; Biological Assay - standards ; Biomarkers ; Biomarkers - blood ; Clinical outcomes ; Colorectal cancer ; Female ; Gene Expression Profiling ; Humans ; Informatics ; Information science ; International conferences ; Lung cancer ; Male ; Medicine ; Methods ; Microfluidics ; MicroRNA ; MicroRNAs ; MicroRNAs - blood ; MicroRNAs - genetics ; MicroRNAs - isolation & purification ; Middle Aged ; miRNA ; Neoplasms ; Nucleic acid isolation ; Nucleic acids ; Parameters ; Patients ; Phlebotomy ; Physiological aspects ; Plasma ; Polymerase chain reaction ; Public health ; Reproducibility ; Reproducibility of Results ; Ribonucleic acid ; RNA ; RNA - genetics ; RNA - isolation & purification ; Separation ; Statistical analysis ; Statistical analysis of data ; Statistical methods ; Surgery ; Systematic review ; Tumors
É parte de: PloS one, 2015-04, Vol.10 (4), p.e0121948-e0121948
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
Competing Interests: The authors have declared that no competing interests exist. The authors also have no commercial interest or support from any manufacturer or industry to disclose.
Conceived and designed the experiments: JR HR SG. Performed the experiments: JR HR JB VS. Analyzed the data: SNR JP JR JB HR VS SG. Contributed reagents/materials/analysis tools: JR HR SG. Wrote the paper: JR SNR SG. Statistical analysis: SNR JP.
Descrição: There are increasing reports of plasma miRNAs as biomarkers of human disease but few standards in methodologic reporting, leading to inconsistent data. We systematically reviewed plasma miRNA studies published between July 2013-June 2014 to assess methodology. Six parameters were investigated: time to plasma extraction, methods of RNA extraction, type of miRNA, quantification, cycle threshold (Ct) setting, and methods of statistical analysis. We compared these data with a proposed standard methodologic technique. Beginning with initial screening for 380 miRNAs using microfluidic array technology and validation in an additional cohort of patients, we compared 11 miRNAs that exhibited differential expression between 16 patients with benign colorectal neoplasms (advanced adenomas) and 16 patients without any neoplasm (controls). Plasma was isolated immediately, 12, 24, 48, or 72 h following phlebotomy. miRNA was extracted using two different techniques (Trizol LS with pre-amplification or modified miRNeasy). We performed Taqman-based RT-PCR assays for the 11 miRNAs with subsequent analyses using a variable Ct setting or a fixed Ct set at 0.01, 0.03, 0.05, or 0.5. Assays were performed in duplicate by two different operators. RNU6 was the internal reference. Systematic review yielded 74 manuscripts meeting inclusion criteria. One manuscript (1.4%) documented all 6 methodological parameters, while < 5% of studies listed Ct setting. In our proposed standard technique, plasma extraction ≤12 h provided consistent ΔCt. miRNeasy extraction yielded higher miRNA concentrations and fewer non-expressed miRNAs compared to Trizol LS (1/704 miRNAs [0.14%] vs 109/704 miRNAs [15%], not expressed, respectively). A fixed Ct bar setting of 0.03 yielded the most reproducible data, provided that <10% miRNA were non-expressed. There was no significant intra-operator variability. There was significant inter-operator variation using Trizol LS extraction, while this was negligible using modified miRNeasy. For standardized reporting, we recommend plasma extraction ≤ 12 h, using modified miRNeasy extraction and utilizing a 0.03 Ct.
Editor: United States: Public Library of Science
Idioma: Inglês

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Assay reproducibility in clinical studies of plasma miRNA

Rice, Jonathan ; Roberts, Henry ; Burton, James ; Pan, Jianmin ; States, Vanessa ; Rai, Shesh N ; Galandiuk, Susan Hoheisel, Jörg D.

United States: Public Library of Science

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