RESUMO
BACKGROUND: MicroRNAs (miRNA) are expected as useful biomarkers for various diseases. We studied the pre-analytical factors causing variation in the analysis of miRNA. MATERIAL AND METHODS: Blood samples were collected from 25 healthy subjects. Plasma and serum were obtained from the same samples. The levels of miR-451, -16, -126, and -223 were analyzed using RT-qPCR. Cel-miR-39 was added as a spiked-in control in each sample. RESULTS: With the exception of miR-451, the levels of the miRNAs in plasma were higher than in serum. After high-speed centrifugation, the levels of miRNAs were almost equal between plasma and serum except for miR-451. Membrane filtration with 0.45 µm pore size reduced the levels of plasma miRNAs. The coagulation accelerators for serum processing did not affect the analysis of miRNA. The use of fraction containing particles of > 0.45 µm in size showed the inhibitory effect on the analysis of plasma miR-451. The RNase inhibitor was effective for protecting against the degradation of miRNAs. CONCLUSION: Plasma contains factors modifying miRNA profiles. The immediate processing of plasma with membrane filtration and RNase inhibitor may be a relevant method for achieving the stable analysis of miRNA.
Assuntos
Biomarcadores/análise , Coleta de Amostras Sanguíneas/normas , MicroRNA Circulante/análise , MicroRNA Circulante/genética , Plasma/química , Soro/química , Adulto , Feminino , Voluntários Saudáveis , Humanos , Masculino , Plasma/metabolismo , Controle de Qualidade , Soro/metabolismo , Adulto JovemRESUMO
MicroRNAs (miRNA) are non-coding small RNAs. Exosomes carry extracellular miRNAs in plasma and other body fluids. Levels of plasma miRNAs show disease-specific changes. Thus, miRNAs are expected to be new biomarkers in many diseases. However, the method of analysis of plasma miRNAs is not well established. In this study, we tested the influences of high speed centrifugation and membrane filtration on results from plasma miRNA analysis using reverse transcriptase-based quantitative polymerase chain reac- tion (RT-qPCR). We studied plasma from 12 normal subjects. The level of plasma miR-451 did not change significantly after high speed centrifugation and filtration, rather showed slight increment, 1.543 ± 0.263 fold (mean±SD, N=3). The levels of plasma miR-126 and miR-223 decreased with high speed centrifugation and filtration, (0.038 ± 0.008 fold and 0.041 ± 0.003 fold, respectively). Our data suggested that removing platelets and cellular debris from plasma with high speed centrifugation and/or filtration is essential for stand- ardization of plasma miRNA analysis. [Original].
Assuntos
Biomarcadores , MicroRNAs , Biomarcadores/análise , Centrifugação , MicroRNAs/análise , Plasma , Reação em Cadeia da Polimerase em Tempo Real , Padrões de ReferênciaRESUMO
MicroRNA (miRNA) is an important regulator of cellular proliferation, differentiation and death. Leukemia-specific signature of miRNAs suggests that epigenetic dysregulation of miRNAs is important for leukemogenesis. We focused on the role of DNA methylation of miR-203 which targets BCR-ABL1 mRNA. The microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment. Imatinib induced the demethylation of the miR-203 promoter region, resulting in low expression of targeted BCR-ABL1 gene, and loss of proliferation of leukemic cells. In conclusion, demethylation of miR-203 is one of the molecular mechanisms of imatinib-induced inhibition of BCR-ABL1-positive leukemic cells.
