Circulating miRNAs are protected from ribonuclease degradation by assembly into microvesicles and exosomes. Releasing miRNAs completely from these particles is the key step to quantify the circulating miRNAs. Currently purified RNA-based quantitative analysis is widely used while it is time and cost consuming with high risk for those circulating miRNAs with low abundance due to partial loss of RNA during the steps of total RNA extraction and small RNA enrichment.
Researchers from Tongji University School of Medicine have optimized a simple, effective and time-saving method to directly measure plasma miRNAs without RNA isolation. It is based on complete miRNA release from the protein complexes, followed by miRNA-specific reverse transcription and quantitative real-time PCR amplification. By comparison to the RNA-based approach, the direct quantification method showed more efficiency for circulating miRNA analysis, higher accuracy and specificity. By application of the direct quantification method to clinical samples combined with the RNA-based miRNA screening analysis, upregulation of miR-106a in blood was validated in metastatic breast cancer patients, indicating miR-106a are a potential biomarker for metastatic breast cancer.
Direct quantification of circulating miRNAs using plasma.
(A) Schematic representation of the three methods (M1, M2 and M3) directly analyzing circulating miRNAs from plasma. (B) QRT-PCR analysis of small RNA 5s rRNA from aliquots of same plasma sample using methods M1, M2 and M3, respectively. (C) QRT-PCR analysis of miR-16 from aliquots of same plasma sample using methods M1, M2 and M3, respectively.