High Throughput qPCR Expression Profiling of Circulating MicroRNAs Reveals Minimal Sex- and Sample Timing-Related Variation in Plasma of Healthy Volunteers

Abstract

MicroRNAs are a class of small non-coding RNA that regulate gene expression at a post-transcriptional level. MicroRNAs have been identified in various body fluids under normal conditions and their stability as well as their dysregulation in disease opens up a new field for biomarker study. However, diurnal and day-to-day variation in plasma microRNA levels, and differential regulation between males and females, may affect biomarker stability. A QuantStudio 12K Flex Real-Time PCR System was used to profile plasma microRNA levels using OpenArray in male and female healthy volunteers, in the morning and afternoon, and at four time points over a one month period. Using this system we were able to run four OpenArray plates in a single run, the equivalent of 32 traditional 384-well qPCR plates or 12,000 data points. Up to 754 microRNAs can be identified in a single plasma sample in under two hours. 108 individual microRNAs were identified in at least 80% of all our samples which compares favourably with other reports of microRNA profiles in serum or plasma in healthy adults. Many of these microRNAs, including miR-16-5p, miR-17-5p, miR-19a-3p, miR-24-3p, miR-30c-5p, miR-191-5p, miR-223-3p and miR-451a are highly expressed and consistent with previous studies using other platforms. Overall, microRNA levels were very consistent between individuals, males and females, and time points and we did not detect significant differences in levels of microRNAs. These results suggest the suitability of this platform for microRNA profiling and biomarker discovery and suggest minimal confounding influence of sex or sample timing. However, the platform has not been subjected to rigorous validation which must be demonstrated in future biomarker studies where large differences may exist between disease and control samples.

Circulating Brain‐Enriched MicroRNAs for Detection and Discrimination of Idiopathic and Genetic Parkinson's Disease

“… and can be detected in all peripheral fluids including saliva, serum, plasma, cerebral spinal fluid (CSF), and urine . Compared to protein biomarkers, miRNAs offer several advantages: (1) they are stable in bodily fluids; (2) they do not get modified; (3) many have restricted expression in the tissues they regulate; (4) preliminary studies indicate little variation between sexes, age, or time of sampling; and (5) they are easily and accurately quantified by routine and fast laboratory methods (such as reverse transcription polymerase chain reaction) that are available in most clinical laboratory settings. With respect to PD, the expression levels of many miRNAs are altered in the different regions of PD brain (reviewed in refs.…”

miRNAs Differentially Expressed by Next-Generation Sequencing in Cord Blood Buffy Coat Samples of Boys and Girls

“…One small study (n = 18) reported 40 miRNAs that were differentially expressed by sex in the human prefrontal cortex [20]. A few studies have examined sex-related differences in profiles of circulating plasma miRNAs in adults but were limited either by small sample size or low number of miRNAs assessed [36][37][38][39]. For example, one study found no differences by sex in miRNA expression of 108 miRNAs characterized in 20 adults [39].…”

“…A few studies have examined sex-related differences in profiles of circulating plasma miRNAs in adults but were limited either by small sample size or low number of miRNAs assessed [36][37][38][39]. For example, one study found no differences by sex in miRNA expression of 108 miRNAs characterized in 20 adults [39]. In another human study, (n = 18) four miRNAs (out of 534 miRNAs) were significantly upregulated in women [38], while yet another study reported seven out of 179 candidate miRNAs to be differentially expressed by sex [36].…”

Standardization procedure for plasma biomarker analysis in rat models of epileptogenesis: Focus on circulating microRNAs

“…Absorbance of hemoglobin in some samples indicated values >0.25 (Fig. C), which was set as an upper limit …”

“…An alternative method is to measure hemoglobin at 414 nm and use a cutoff absorbance value of 0.20-0.25. [18][19][20] Although not a typical problem in rat samples, it should be noted that lipemia in plasma interferes with hemoglobin absorbance, which may cause an increase in absorbance values, even in the absence of any hemoglobinrelated peak. 21 By also measuring the plasma absorbance at 385 nm (as a lipemia indicator) it is possible to identify hemolyzed samples, independent of lipemia.…”