[Standardization of Plasma Preparation for MicroRNA Analysis: Influences of High Speed Centrifugation and Membrane Filtration].

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].

Serum microRNAs as new biomarkers for detecting subclinical hemolysis in the nonacute phase of G6PD deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common enzymopathies worldwide. Patients with G6PD deficiency are usually asymptomatic throughout their life but can develop acute hemolysis after exposure to free radicals or certain medications. Several studies have shown that serum miRNAs can be used as prognostic biomarkers in various types of hemolytic anemias. However, the impact of G6PD deficiency on circulating miRNA profiles is largely unknown. The present study aimed to assess the use of serum miRNAs as biomarkers for detecting hemolysis in the nonacute phase of G6PD deficiency. Patients with severe or moderate G6PD Viangchan (871G > A) deficiency and normal G6PD patients were enrolled in the present study. The biochemical hemolysis indices were normal in the three groups, while the levels of serum miR-451a, miR-16, and miR-155 were significantly increased in patients with severe G6PD deficiency. In addition, 3D analysis of a set of three miRNAs (miR-451a, miR-16, and miR-155) was able to differentiate G6PD-deficient individuals from healthy individuals, suggesting that these three miRNAs may serve as potential biomarkers for patients in the nonhemolytic phase of G6PD deficiency. In conclusion, miRNAs can be utilized as additional biomarkers to detect hemolysis in the nonacute phase of G6PD deficiency.

Pre-Analytical Modification of Serum miRNAs: Diagnostic Reliability of Serum miRNAs in Hemolytic Diseases.

Circulating microRNAs (miRNAs) are useful biomarkers of hemolysis. Since blood cells are the main origins of circulating miRNAs, we evaluated blood cell-related pre-analytical modification of the miRNA signatures during blood drawing and serum processing. The levels of miRNA before and after ex vivo blood drawing were analyzed with the reverse transcriptase-based polymerase chain reaction method. Furthermore, the changes of miRNA signatures caused by different time-lag between blood drawing and serum preparation by 24 h were evaluated. Finally, we compared the miRNA levels between leftover samples and samples of hemolytic diseases. Blood drawing procedure induced increments of red blood cell (RBC)-related miRNAs (miR-451a, miR-486) about 2-fold. One hour standing of blood samples before serum separation induced almost the same increases in RBC-related miRNAs. To test the clinical usefulness of miR-451a as a biomarker of hemolytic diseases, we analyzed miRNAs of samples from 10 normal subjects, 30 leftover samples in the clinical laboratory, and 20 samples from patients with hemolytic diseases. Serum miR-451a significantly increased in patients with hemolytic anemia more than the levels of pre-analytical modification. In conclusion, the pre-analytical modification of serum miRNAs did not disturb the usefulness of RBC-derived miRNAs as biomarkers of hemolytic diseases.

The Influence of Pre-Analytical Factors on the Analysis of Circulating MicroRNA.

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.

Upregulation of microRNA-31 associates with a poor prognosis of malignant pleural mesothelioma with sarcomatoid component.

Malignant pleural mesothelioma (MPM) is a neoplastic disease with a poor prognosis. The complete resection of tumor with extra-pleural pneumonectomy is effective only for early stage epithelioid-type MPM. An accurate pathological diagnosis including the histological subtype and also clinical staging are crucial to decide on the therapeutic approach. However, the pathological diagnosis is difficult when the amount of biopsy sample is small. We performed a comparative analysis of the miR-31 expression in MPM and reactive mesothelial proliferations (RMPs), by RT-qPCR of formaldehyde-fixed paraffin-embedded samples, and compared the expression levels of miR-31 with the results of a survival analysis for the diagnosis and prognosis of MPM. The expression of miR-31 was found to be significantly reduced in MPMs compared with RMPs (P < 0.01). The pathological subtype of four among five cases with upregulated miR-31 levels was MPM with a sarcomatoid component. (i.e., biphasic or sarcomatoid type). Furthermore, these four cases were significantly associated with a worse prognosis compared with the five cases of biphasic or sarcomatoid MPM without upregulated miR-31 expression (P = 0.0027). In conclusion, the analysis of miR-31 expression levels may be a good biomarker for diagnosis of MPM histological typing and predicting the prognosis.

Imatinib induces demethylation of miR-203 gene: an epigenetic mechanism of anti-tumor effect of imatinib.

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.