Plasma microrna quantification protocol

Abstract

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.

Keywords: MicroRNA; aortic aneurysm; ddPCR; plasma; quantification.

Figure 1.

Preparation and Serial Dilution of Exogenous Spike-in Control. (A) Serial dilution instructions for creating a 25 μM stock solution of cel-miR-39–3p (miR-39) spike-in. First, a 0.5 μM solution of miR-39 spike-in is prepared by performing a 1:50 dilution in water. The actual stock concentration (in ng/μL) is then measured by a Qubit Fluorometer and microRNA Concentration Kit. The concentration of the stock solution is adjusted to 1,000 ng/μL. Serial dilutions of the miR-39 spike-in are prepared in water as shown. All dilutions are prepared by mixing 1 part of miR-39 spike-in solution with 9 parts water, vortexing (30 s) and resting on ice (30 s) 5 times before subsequent dilution. (B) Representative 1-Demensional analysis of 01–0.0001 ng/μL and the respective No Template Control (NTC). The green dots represent VIC positive (miR-39) particles, while the grey dots represent droplets with no fluorescence. (C) Ratio of miR-39 positive to total events. Results represent triplicate measurements, ±standard error of the mean (SEM). (D) Measured concentrations of miR-39 (copy number/μL), where the solid line is the median, and the bar represents the interquartile range.

Figure 2.

Schematic diagram of the microRNA quantification workflow. (A) Blood collection, plasma isolation, and long-term storage. Peripheral venous blood is collected in BD Vacutainer^® EDTA-coated tube and plasma is separated by centrifugation (2,500× g, 15 min, room temperature). Plasma supernatant is removed and centrifuged again and supernatant is aliquoted for storage at −80 °C. (B) Small RNA Isolation from Plasma. microRNA is isolated according to the manufacturer’s instructions of the Qiagen miRNeasy Serum/Plasma Advanced Kit. (C) Small RNA quantification and concentration normalization. The concentration (ng/μL) of eluted microRNA is quantified using the Qubit microRNA assay, according to manufacturer’s instructions. Concentration is subsequently adjusted to 122.55 ng/μL for all samples. (D) Spike-in addition and microRNA specific cDNA generation. miR-39 is exogenously spiked into the microRNA sample, and target-specific cDNA generation is performed utilizing TaqMan microRNA Reverse Transcription Kit following manufacturer’s instructions. miR-39-specific and target-specific cDNA generation are performed separately to avoid cross-reactivity. (E) Droplet generation and microRNA specific PCR amplification. miR-39 cDNA and target-specific cDNA are mixed together with ddPCR supermix (no DUTP), and respective FAM and VIC miR-39 and target-specific cDNA amplification primers. Samples are transferred to Bio-Rad droplet generator microfluidic chips alongside droplet generator oil and transferred to the QX200 Droplet Generator. Droplets are transferred to a deep-well PCR plate, followed by PCR amplification. (F) Droplet reading and data analysis. The samples are read by the Bio-Rad QX200 Droplet Reader and analyzed with Bio-Rad QuantaSoft software. All appropriate instructions in user manuals are adhered to.

Figure 3.

Expected range of microRNA concentration following isolation. microRNA is isolated from plasma with the Qiagen miRNeasy Serum/Plasma Advanced Kit, and the concentration of microRNA is quantified by the Qubit microRNA assay. Results represent 47 independent biological samples where the dotted line is the mean, and the solid line is the median, and the bar represents the interquartile range.

Figure 4.

Representative 2-dimensional ddPCR plots of cardiovascular disease related microRNAs. ddPCR plots are separated into 4 quadrants representing FAM(−), VIC(−) events (lower left), FAM(−), VIC(+) events (lower right), FAM(+), VIC(−) events (upper left), and FAM(+) VIC(+) events (upper right). The analysis allows for quantification of total copy numbers, as well as a ratio of the microRNA of interest specific events [FAM(+), VIC(+)] normalized to the spike-in of miR39, represented by FAM(−), VIC(+) events.