Human plasma and serum extracellular small RNA reference profiles and their clinical utility

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell–specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell–specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies

Cell and Microvesicle Urine microRNA Deep Sequencing Profiles from Healthy Individuals: Observations with Potential Impact on Biomarker Studies.

Urine is a potential source of biomarkers for diseases of the kidneys and urinary tract. RNA, including microRNA, is present in the urine enclosed in detached cells or in extracellular vesicles (EVs) or bound and protected by extracellular proteins. Detection of cell- and disease-specific microRNA in urine may aid early diagnosis of organ-specific pathology. In this study, we applied barcoded deep sequencing to profile microRNAs in urine of healthy volunteers, and characterized the effects of sex, urine fraction (cells vs. EVs) and repeated voids by the same individuals.Compared to urine-cell-derived small RNA libraries, urine-EV-derived libraries were relatively enriched with miRNA, and accordingly had lesser content of other small RNA such as rRNA, tRNA and sn/snoRNA. Unsupervised clustering of specimens in relation to miRNA expression levels showed prominent bundling by specimen type (urine cells or EVs) and by sex, as well as a tendency of repeated (first and second void) samples to neighbor closely. Likewise, miRNA profile correlations between void repeats, as well as fraction counterparts (cells and EVs from the same specimen) were distinctly higher than correlations between miRNA profiles overall. Differential miRNA expression by sex was similar in cells and EVs.miRNA profiling of both urine EVs and sediment cells can convey biologically important differences between individuals. However, to be useful as urine biomarkers, careful consideration is needed for biofluid fractionation and sex-specific analysis, while the time of voiding appears to be less important

Comparison of Systolic Blood Pressure Values Obtained by Photoplethysmography and by Korotkoff Sounds

In the current study, a non-invasive technique for systolic blood pressure (SBP) measurement based on the detection of photoplethysmographic (PPG) pulses during pressure-cuff deflation was compared to sphygmomanometry—the Korotkoff sounds technique. The PPG pulses disappear for cuff-pressures above the SBP value and reappear when the cuff-pressure decreases below the SBP value. One hundred and twenty examinations were performed on forty subjects. In 97 examinations the two methods differed by less than 3 mmHg. In nine examinations the SBP value measured by PPG was higher than that measured by sphygmomanometry by 5 mmHg or more. In only one examination the former was lower by 5 mmHg or more than the latter. The appearance of either the PPG pulses or the Korotkoff sounds assures that the artery under the cuff is open during systolic peak pressure. In the nine examinations mentioned above the PPG pulses were observed while Korotkoff sounds were not detected, despite the open artery during systole. In these examinations, the PPG-based technique was more reliable than sphygmomanometry. The high signal-to-noise ratio of measured PPG pulses indicates that automatic measurement of the SBP by means of automatic detection of the PPG signals is feasible