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Circulating cell-free nucleic acids (NAs), in particular plasma-derived cell-free DNA, have evolved into promising clinical analytes for prenatal diagnostics, cancer analysis, and cancer surveillance and therapy monitoring. Nevertheless, salivary extracellular and extracellular vesicle (EV)-derived DNA and microRNA have recently gained attention as potential non-invasive biomarkers for a variety of diseases, including cancer, cardiovascular, autoimmune, and infectious diseases. Our goal in this study was therefore to evaluate and optimize commercially available approaches for cell-free nucleic acid isolation, focusing specifically on DNA and miRNA present in cell-free saliva or saliva-derived EVs. Along these lines, we investigated various commercially available kits, which enable parallel isolation of cell-free DNA and RNA in separate fractions from cell-free saliva and salivary EVs, respectively, and compared them to single analyte extraction kits. The efficiency of all tested nucleic acid extraction methods was determined by comparing DNA and RNA fluorescence spectroscopy measurements and quantitative PCR values obtained from a selection of different DNA- and microRNA targets. We found the Norgen Plasma/Serum RNA/DNA Purification Mini kit in combination with the miRCURY exosome isolation kit to work best in our hands and to provide the highest yields of EV-derived nucleic acids. Having tested and identified effective protocols for isolating salivary extracellular nucleic acids, we present with this comparison study, among others, a sound basis for future circulating small nucleic acid and epigenetic biomarker research aiming for early disease diagnosis, prognosis, and prediction from cell-free saliva, representing an easy-to-collect and readily available diagnostic fluid.

MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non-vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2-miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.

The aim of this study was to identify microRNAs in urinary exosomes that are differently expressed in prostate cancer patients and healthy donors. For this purpose, RNA was extracted from urinary exosomes from 20 prostate cancer patients and 9 healthy males and the microRNAs were analyzed by next generation sequencing. Interestingly, 5 microRNAs – miR-196a-5p, miR-34a-5p, miR-143-3p, miR-501-3p and miR-92a-1-5p – were significantly downregulated in exosomes from prostate cancer patients. Furthermore, RT-qPCR analysis of an independent cohort of 28 prostate cancer patients and 19 healthy males confirmed that miR-196a-5p and miR-501-3p were downregulated in prostate cancer samples. These results suggest that specific microRNAs in urinary exosomes might serve as non-invasive biomarkers for prostate cancer. In particular, miR-196a-5p and miR-501-3p are promising biomarkers that need to be further studied in large patient cohorts.

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate various biological processes, primarily through interaction with messenger RNAs. The levels of specific, circulating miRNAs in blood have been shown to associate with various pathological conditions including cancers. These miRNAs have great potential as biomarkers for various pathophysiological conditions. In this study we focused on different sample types' effects on the spectrum of circulating miRNA in blood. Using serum and corresponding plasma samples from the same individuals, we observed higher miRNA concentrations in serum samples compared to the corresponding plasma samples. The difference between serum and plasma miRNA concentration showed some associations with miRNA from platelets, which may indicate that the coagulation process may affect the spectrum of extracellular miRNA in blood. Several miRNAs also showed platform dependent variations in measurements. Our results suggest that there are a number of factors that might affect the measurement of circulating miRNA concentration. Caution must be taken when comparing miRNA data generated from different sample types or measurement platforms.

MicroRNAs (miRNAs) have emerged as important regulators in the post‐transcriptional control of gene expression. The discovery of their presence not only in tissues but also in extratissular fluids, including blood, urine and cerebro‐spinal fluid, together with their changes in expression in various pathological conditions, has implicated these extracellular miRNAs as informative biomarkers of disease. However, exploiting miRNAs in this capacity requires methodological rigour. Here, we report several key procedural aspects of miRNA isolation from plasma and serum, as exemplified by research in cardiovascular and pulmonary diseases. We also highlight the advantages and disadvantages of various profiling methods to determine the expression levels of plasma‐ and serum‐derived miRNAs. Attention to such methodological details is critical, as circulating miRNAs become diagnostic tools for various human diseases.

MicroRNA exhibits differential expression levels in cancer and can affect cellular transformation, carcinogenesis and metastasis. Although fluorescence techniques using dye molecule labels have been studied, label-free molecular-level quantification of miRNA is extremely challenging. We developed a surface plasmon resonance sensor based on two-dimensional nanomaterial of antimonene for the specific label-free detection of clinically relevant biomarkers such as miRNA-21 and miRNA-155. First-principles energetic calculations reveal that antimonene has substantially stronger interaction with ssDNA than the graphene that has been previously used in DNA molecule sensing, due to thanking for more delocalized 5 s /5 p orbitals in antimonene. The detection limit can reach 10 aM, which is 2.3–10,000 times higher than those of existing miRNA sensors. The combination of not-attempted-before exotic sensing material and SPR architecture represents an approach to unlocking the ultrasensitive detection of miRNA and DNA and provides a promising avenue for the early diagnosis, staging, and monitoring of cancer. Label-free molecular-level quantification of MicroRNA (miRNA) remains challenging. Here, the authors develop a new surface plasmon resonance sensor based on two-dimensional nanomaterial of antimonene for the specific label-free detection of clinically relevant biomarkers such as miRNA-21 and miRNA-155.

Background Breast cancer patients under neoadjuvant chemotherapy includes a heterogeneous group of patients who eventually develop distal disease, not detectable by current methods. We propose the use of exosomal miRNAs and circulating tumor cells as diagnostic and predictive biomarkers in these patients. Methods Fifty-three breast cancer women initially diagnosed with localized breast cancer under neoadjuvant chemotherapy were prospectively enrolled in this study. However, six of them were later re-evaluated and diagnosed as metastatic breast cancer patients by PET-CT scan. Additionally, eight healthy donors were included. Circulating tumor cells and serum exosomal miRNAs were isolated from blood samples before and at the middle of neoadjuvant therapy and exosomal miRNA levels analyzed by qPCR. Results Before neoadjuvant therapy, exosomal miRNA-21 and 105 expression levels were higher in metastatic versus non-metastatic patients and healthy donors. Likewise, higher levels of miRNA-222 were observed in basal-like ( p  = 0.037) and in luminal B versus luminal A ( p  = 0.0145) tumor subtypes. Exosomal miRNA-222 levels correlated with clinical and pathological variables such as progesterone receptor status ( p  = 0.017) and Ki67 ( p  = 0.05). During neoadjuvant treatment, exosomal miRNA-21 expression levels directly correlated with tumor size ( p  = 0.039) and inversely with Ki67 expression ( p  = 0.031). Finally, higher levels of exosomal miRNA-21, miRNA-222, and miRNA-155 were significantly associated with the presence of circulating tumor cells. Conclusion Liquid biopsies based on exosomal miRNAs and circulating tumor cells can be a complementary clinical tool for improving breast cancer diagnosis and prognosis.

MicroRNAs (miRNAs) are promising biomarkers for the early detection of various diseases, particularly cancer, driving active development of highly sensitive and selective detection technologies. This study aims to establish a novel miRNA detection technique that utilizes image analysis to track the Brownian motion (diffusivity) of fluorescent probe-modified miRNA particles. This method identifies the presence and concentration of miRNAs by exploiting the change in particle size upon hybridization with the target. Furthermore, the use of a probe modified with a photo-crosslinkable artificial nucleic acid (CNV-D) enables the covalent capture of the target miRNA, ensuring high selectivity in biological samples even under stringent washing conditions. By integrating Hybridization Chain Reaction (HCR), the complex size is significantly amplified, dramatically enhancing the detection sensitivity. Consequently, we successfully demonstrated the highly sensitive and specific detection of the cancer biomarker miR-21 in serum, achieving an exceptionally low limit of detection (LOD) of 1 fM. This technology holds great potential to contribute to the early diagnosis of cancer.

MicroRNAs (miRNAs) are short (19–24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. Differential expression profiles of miRNAs across a range of diseases have emerged as powerful biomarkers, making a reliable yet rapid profiling technique for miRNAs potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Compared to previously reported techniques, our technique can discriminate single base mismatches and single-nucleotide 3′-tailing with low false positive rates regardless of their positions on miRNA. By preloading probes in Thermus thermophilus Argonaute ( Tt Ago), miRNAs detection speed is accelerated by more than 20 times. Finally, by utilizing the well-conserved linearity between single-molecule spot numbers and the target miRNA concentrations, the absolute average copy numbers of endogenous miRNA species in a single cell can be estimated. Thus our technique, Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip. MicroRNAs are potentially powerful biomarkers, though clinical use requires rapid and reliable profiling. Here the authors report amplification-free multicolour single-molecule imaging with single base mismatch sensitivity.

Background Circulating microRNAs (miRNAs) are attractive non-invasive biomarkers for a variety of conditions due to their stability and altered pathophysiological expression levels. Reliable detection of global expression profiles is required to maximise miRNA biomarker discovery. Although developments in small RNA-Seq technology have improved detection of plasma-based miRNAs, the low RNA content and sequencing bias introduced during library preparation remain challenging. In this study we compare commercially available RNA extraction methods using MagnaZol (Bioo Scientific) or miRNeasy (QIAGEN) and three library preparation methods - CleanTag (TriLink), NEXTflex (Bioo Scientific) and QIAseq (QIAGEN) - which aim to address one or both of these issues. Results Different RNA extractions and library preparation protocols result in differential detection of miRNAs. A greater proportion of reads mapped to miRNAs in libraries prepared with MagnaZol RNA than with miRNeasy RNA. Libraries prepared using QIAseq demonstrated the greatest miRNA diversity with many more very low abundance miRNAs detected (~ 2–3 fold more with < 10 reads), whilst CleanTag detected the fewest individual miRNAs and considerably over-represented miR-486-5p. Libraries prepared with QIAseq had the strongest correlation with RT-qPCR quantification. Analysis of unique molecular indices (UMIs) incorporated in the QIAseq protocol indicate that little PCR bias is introduced during small RNA library preparation. Conclusions Small RNAs were consistently detected using all RNA extraction and library preparation protocols tested, but with some miRNAs at significantly different levels. Choice of the most suitable protocol should be informed by the relative importance of minimising the total sequencing required, detection of rare miRNAs or absolute quantification.