[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].

Downregulation of plasma miR-451 and miR-16 in Plasmodium vivax infection.

Malaria is a common parasitic disease in tropical countries, causing one to two million deaths every year. To establish the new biomarker, we analyzed plasma miRNAs obtained from 19 malaria patients and 19 normal subjects, using reverse transcription-based quantitative polymerase chain reaction (RT-qPCR). The average levels of plasma miR-451 and miR-16 were significantly lower in malaria patients, (8.9-fold; p <0.001 and 10.4-fold; p = 0.01, respectively). The levels of other abundant miRNAs in plasma (miR-223, miR-226-3p) did not change significantly in malaria patients. Our data suggest that plasma miR-451 and miR-16 are relevant biomarkers for malaria infection.

[Circulating MicroRNAs as Biomarkers of Colorectal Cancer].

Colorectal cancer is a common tumor in Japan, causing almost 50,000 deaths per year. The development of new biomarkers is strongly desired, in order to detect the early stage of colorectal cancer with high sensitivity and specificity, using less invasive and high through-put methods. miRNA is a small non-coding RNA which regulates gene expression by digesting mRNA or suppressing translation. miRNAs are stable and present in blood, urine, stool, and other body fluids. The profiles of miRNAs in body fluid are specific to pathological states. There is accumulating data showing the usefulness of miRNAs as new biomarkers for colorectal cancer. We summarize the current knowledge in the previous literature (10 plasma analyses: sensitivity: 83.3 to 89%, specificity: 41 to 84.7%, AUC: 0.606 to 0.896; 13 serum analyses: sensitivity: 66.7 to 96.4%, specificity: 63.9 to 88.1%, AUC: 0.679 to 0.918; and 8 fecal analyses: sensitivity: 70.9 to 81.8%, specificity: 68.4 to 96.3%, AUC: 0.64 to 0.829). We focus on the standardization of miRNA analysis, namely: 1) preanalytical processes: difference of miRNA levels between plasma and serum, sampling methods, preparation of plasma or serum, and preservation of samples; 2) analytical processes: mRNA extraction methods, amplification, normalizer, and cut-off values. In conclusion, miRNAs are expected to become new biomarkers for colorectal cancer screening.

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.