Successful treatment of cancer depends on early diagnosis and effective monitoring of patients' response to therapy. Traditional tools based on tumor biopsies lack the sensitivity and specificity to capture cancer development in its early phases and are not applicable for continuous monitoring. To overcome these barriers, liquid biopsies have been introduced as a minimally invasive and cost-efficient means of diagnosis with high level of specificity and sensitivity. Traditionally, liquid biopsy markers include circulating tumor cells and circulating tumor DNA. During the last decade, a new promising group of biomarkers has appeared and its utilization for cancer diagnosis and monitoring is intensively studied - the microRNAs (miRNAs). In this review, we provide a comprehensive overview of circulating miRNA analysis. We highlight the importance of sampling and quality control, discuss the technical aspects of miRNA extraction and quantification, summarize recommendations for downstream analysis and conclude with future perspectives. Taken together, we present the current state of knowledge in the field of miRNA analysis in liquid biopsies and the expected development and standardization.

• MeSH
• Circulating MicroRNA analysis   isolation & purification   MeSH
• Humans MeSH
• Biomarkers, Tumor genetics   MeSH
• Neoplasms diagnosis   genetics   therapy   MeSH
• Specimen Handling methods   MeSH
• Quality Control MeSH
• Liquid Biopsy MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.

• MeSH
• Caenorhabditis elegans chemistry   MeSH
• Chemical Fractionation methods   MeSH
• Circulating MicroRNA blood   isolation & purification   MeSH
• Extracellular Vesicles chemistry   MeSH
• Real-Time Polymerase Chain Reaction methods   MeSH
• Middle Aged MeSH
• Humans MeSH
• Biomarkers, Tumor blood   isolation & purification   MeSH
• Reverse Transcriptase Polymerase Chain Reaction methods   MeSH
• Aged MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Animals MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH

Circulating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts invested in the field, the research so far has failed to deliver expected results. One of the contributing factors is general lack of agreement between various studies, partly due to the considerable technical challenges accompanying the workflow. Pre-analytical variables including sample collection, RNA isolation, and quantification are sources of bias that may hamper biological interpretation of the results. Here, we present a Two-tailed RT-qPCR panel for quality control, monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The Two-tailed QC (quality control) panel is based on two sets of synthetic spike-in molecules and three endogenous microRNAs that are quantified with the highly specific Two-tailed RT-qPCR technology. The QC panel is a cost-effective way to assess quality of isolated microRNA, degree of inhibition, and erythrocyte contamination to ensure technical soundness of the obtained results. We provide assay sequences, detailed experimental protocol and guide to data interpretation. The application of the QC panel is demonstrated on the optimization of RNA isolation from biofluids with the miRNeasy Serum/Plasma Advanced Kit (Qiagen).

• MeSH
• Cost-Benefit Analysis MeSH
• Biomarkers blood   MeSH
• Circulating MicroRNA blood   isolation & purification   MeSH
• Rats MeSH
• Real-Time Polymerase Chain Reaction economics   instrumentation   methods   standards   MeSH
• Humans MeSH
• Reagent Kits, Diagnostic standards   MeSH
• Quality Control * MeSH
• Feasibility Studies MeSH
• Healthy Volunteers MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cell-free microRNAs (miRNAs) have become one of the novel promising diagnostic and prognostic biomarkers for various diseases recently. Blood serum and plasma along with urine are the most common sources of clinically well, almost noninvasively available samples containing various types of miRNAs. Here, we present a protocol for a small-scale study investigating expression of several candidate miRNAs. Small-scale experiments may be worth investigating in cases where no information is available on miRNAs expression in particular diseases, for validation of previously published miRNAs with promising diagnostic potential, particularly in situations where follow-up study is aimed at validating miRNAs coming from array or NGS experiments, or where funding for these large-scale experiments is not available.Using urine miRNAs expression as the novel diagnostic tools is challenging and currently this approach is still in its infancy. Therefore, various methods may result in different conclusions depending on clinical sample sets and differences among methods used for the miRNAs isolation and quantitation. In this protocol, we present the method evaluated in the study focused on cell-free urinary miRNAs in ovarian and endometrial cancers. We recommend using stabilization tubes for the urine collection, as this step may be necessary to stop activity of RNases. Further, routine real-time PCR methods are described. We demonstrate that assessment of urinary miRNAs expression may reveal as a feasible method to explore the potential for finding novel diagnostic and prognostic markers.

• MeSH
• Urinalysis methods   MeSH
• Circulating MicroRNA genetics   isolation & purification   urine   MeSH
• Real-Time Polymerase Chain Reaction methods   MeSH
• Humans MeSH
• Endometrial Neoplasms genetics   urine   MeSH
• Ovarian Neoplasms genetics   urine   MeSH
• Reverse Transcriptase Polymerase Chain Reaction methods   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Gene Expression Profiling methods   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

• MeSH
• Biomarkers MeSH
• Circulating MicroRNA * isolation & purification   classification   blood   MeSH
• Adult MeSH
• Plasma MeSH
• Middle Aged MeSH
• Humans MeSH
• Microarray Analysis MeSH
• Myelodysplastic Syndromes * diagnosis   MeSH
• Polymerase Chain Reaction MeSH
• Prognosis * MeSH
• Disease Progression MeSH
• ROC Curve MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Research MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Circulating MicroRNA genetics   isolation & purification   urine   MeSH
• Exosomes genetics   pathology   MeSH
• Humans MeSH
• Endometrial Neoplasms * diagnosis   urine   pathology   MeSH
• Ovarian Neoplasms * diagnosis   urine   pathology   MeSH
• Pilot Projects MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH