Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Summary

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Abstract

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Introduction

Circulating miRNAs have been identified as promising markers for a number of diseases, including cardiovascular disease¹. The miRNAs are small, non-coding single-stranded RNA molecules (approximately 22 nucleotides long) are involved in the post-transcriptional regulation via the alteration of the messenger RNA translation and influencing gene expression², and are released into the circulation in both physiological and pathological states. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity¹. In cardiovascular diseases, miRNAs have become attractive candidates as novel biomarkers because they are remarkably stable in the serum and can easily be quantified with the help of PCR methodology³. The potential value of miRNAs as biomarkers for myocardial infarction has been evaluated in small studies, but a validation in large cohorts is lacking². For example, miR-499 is found highly expressed in the myocardial muscle, and it has been shown to be significantly increased in an AMI^4,5,6. Further, it regulates programmed cell death (apoptosis) and the differentiation of cardiomyocytes and is thus involved in several mechanisms following an AMI⁷. Apart from some small studies reporting a superiority and incremental value of miRNAs for the diagnosis of AMI, the superiority or equality to high-sensitivity cardiac troponins has not yet been proven in large-scale studies^2,5,6,8. More prospective studies in large cohorts are, therefore, needed to assess the potential diagnostic value of miRNAs. Additionally, methods of miRNA quantification need to be optimized and standardized using comparable protocols⁹. Standardized assays may reduce inconsistent results and may help miRNAs to become potential biomarkers for the routine clinical application, as biomarkers need to be quantified in a reproducible way to ensure their clinical applicability.

Recently, dPCR has been introduced as an end-point analysis. It partitions the sample into approximately 20,000 individual reactions¹⁰. The dPCR system then utilizes a mathematical Poisson statistical analysis of fluorescent signals (positive and negative reactions), enabling an absolute quantification without a standard curve¹⁰. When combining dPCR with fluorescent hydrolysis probes, the highly specific direct absolute quantification of miRNAs is made possible. Digital polymerase chain reaction has shown to exhibit superior technical qualities (including a decreased variability, an increased day-to-day reproducibility, a high degree of linearity, and a high sensitivity) for quantifying miRNA levels in the circulation compared to quantitative real-time PCR^10,11. These superior technical qualities might help to mitigate current limitations on using circulating miRNAs as biomarkers and may lead to the establishment of miRNAs as biomarkers in large multi-center cardiovascular clinical trials and as a diagnostic method in general. In a previous study, we recently applied dPCR for the absolute quantification of circulating miRNAs in patients with an AMI and were able to demonstrate superior diagnostic potential compared to the quantification of miRNAs by qPCR¹².

In this publication, we want to demonstrate that using dPCR is an accurate and reproducible method for directly quantifying circulating cardiovascular miRNAs. The absolute quantification of miRNA levels in serum, using digital PCR, shows potential for the use in large multi-center cardiovascular clinical trials. In this publication, we describe in detail how to effectively perform digital PCR and how to detect the absolute miRNA copy number in serum.

Protocol

1. Extraction of miRNA from Plasma/Serum

Note: In order to quantify miRNAs appropriately, the correct microRNA isolation from the plasma/serum is a crucial step. A key thing to keep in mind, especially because different protocols exist, is to adhere to the same workflow while processing the samples. In this protocol, miRNA is extracted from 50 µL of serum. Do not use more than 200 µL as this limit the correct extraction process.

1. Prepare serum or thaw frozen samples.
2. Add 250 µL (5 volumes) commercial lysis reagent to 50 µL of serum. Support the lysis by vortexing. Place the tube with the lysate on the bench at room temperature for 5 min.
3. Spike the lysate with 3.5 µL of spike-in control (1.6 x 10⁷ copies/µL) and mix them by vortexing.
4. Add 50 µL of chloroform (i.e., an equal amount as to the starting serum sample) for the phase separation. Shake the tube vigorously for 15 s. Place the tube on the bench at room temperature for 2 - 3 min.
5. For the phase separation, centrifuge the tube at 12,000 x g at 4 °C for 15 min.
   Note: The samples are now separated into an upper aqueous phase containing the RNA, a white interphase, and a lower, pink organic phase.
6. Transfer the upper aqueous phase (100 µL) containing the RNA into a new tube. Do not transfer any white interphase material as this falsifies the correct RNA count.
7. Add 150 µL (i.e., 1.5x the volume of the transferred sample) of 100% ethanol. Mix the materials by pipetting them up and down. Do not centrifuge them, and quickly move on to the next step.
8. Pipette 250 µL of the sample onto a commercial spin column in a 2 mL collection tube. Close the lid. Centrifuge the column at >8,000 x g at room temperature for 15 s. Discard the flow-through.
9. Add 700 µL of commercial washing buffer number 1 to the spin column. Close the lid and centrifuge the column at >8,000 x g at room temperature for 15 s. Discard the run-through washing buffer.
10. Add 500 µL of commercial washing buffer number 2 to the spin column. Close the lid and centrifuge the column at >8, 000 x g at room temperature for 15 s. Discard the run-through washing buffer.
11. Pipette 500 µL of 80% ethanol to the spin column. Close the lid, centrifuge the column at >8, 000 x g at room temperature for 2 min. Discard the collection tube with the flow-through.
12. Transfer the spin column to a new 2 mL collection tube. Centrifuge the column at full speed with an opened lid to dry the membrane. Discard the collection tube with the flow-through.
13. Transfer the spin column into a new 1.5 mL collection tube. Elute the RNA by applying 30 µL of RNase-free water directly to the center of the membrane. Close the lid. Centrifuge the column at full speed for 1 min.
14. Store the RNA at -70 °C.
    Note: The storage of purified RNA in RNase-free water between -70 °C to -20 °C ensures no degradation of the RNA for 1 year. Following the manufacturer’s protocol, the minimum amount of RNase-free water to dilute the RNA is 10 µL. Using less than 10 µL may insufficiently hydrate the membrane and reduce the total RNA yield.

2. Reverse Transcription

Note: Complementary DNA (cDNA) was synthesized using the following 15 µL reverse transcription (RT) protocol (total reaction volume).

1. Thaw the RT kit on ice. Keep the enzymes in the freezer as long as possible to prevent them from degrading and spin them down before use. Thaw RT primers on ice and spin them down before use.
2. Prepare the master mix as described in Table 1.
3. Combine 10 µL of master mix and 5 µL of extracted RNA per well in a 96 well-plate.
4. Centrifuge the well plate at 2,000 x g at 4 °C for 2 min.
5. Use the thermal cycle protocol described in Table 2 for the reverse transcription.
   Note: As described below, cDNA may directly be processed in the digital PCR machine (see Table of Materials); alternatively, it may be stored at -20 °C.

3. Droplet Generation and Digital PCR

Note: Digital PCR was performed using the following 40 µL protocol.

1. Thaw the commercial dPCR mix, the prepared cDNA, and the PCR primers on ice. Centrifuge them shortly before use.
2. Prepare the master mix as described in Table 3.
3. Add 1.33 μL/well of the RT product and centrifuge it briefly.
4. Pipette 20 μL of the sample into each well of the 8-well cassette (medium row).
   Note: If the 8-well cassette is not completely filled, fill up the remaining wells with non-template controls (NTCs) that omit cDNA (i.e., the RT product produced with water instead of extracted RNA) or dPCR commercial buffer. The NTC serves as a contamination control. Water must not be used in the remaining wells, because that will impair the quality of the droplet formation.
5. Pipette 70 μL of the droplet generation oil for the probes into the oil wells (bottom row) of the 8-well cassette.
6. Place a gasket on top of the 8-well cassette and place the cassette in the droplet generator. Close the droplet generator. Wait until the 3 indicator lights are solid green.
   Note: Droplets are now being generated.
7. Carefully and slowly transfer 40 μL of the droplet-formed sample (top row) into separate wells of a 96-well plate by pipetting.
8. After completing the droplet formation on the samples, seal the PCR plate with a pierceable dPCR foil at 180 °C for 4 s.
9. Place the foil-sealed PCR-plate in the cycler and cycle it as described in Table 4, at a ramp rate of 2.5 °C/s.

4. Droplet Reading and Analyzing

1. Transfer the plate from the thermal cycler to the base of the plate holder. Tighten the PCR plate by placing the top of the plate holder onto the PCR plate.
2. Start the commercial dPCR software.
3. Enter the sample name, the name of the experiment, and the target in the setup. Select the absolute quantification.
4. Start the droplet reading by clicking RUN. Select FAM/HEX or FAM/VIC as detection chemistry when working with hydrolysis probes.
   Note: The commercial dPCR software will then auto-analyze the data.
5. Check the droplet numbers in the results table to ensure a correct droplet generation.
6. Select all wells with the same quantified miRNA and click ANALYZE. Use the 2D blot to mark positive droplets and manually correct the auto-analyzed data (Figure 1).

5. Correct Calculations on the Sample

1. Normalize the sample by multiplying the sample by the normalization factor (NF).
   NF = Median [C. elegans miR-39 measurements]_{all samples}/[C. elegans miR-39]_{given sample}
2. Calculate the final miRNA serum concentration, adjusting for dilution factors (DF).
   [miRNA]_final = [miRNA]_{raw value} x DF_RT x DF_dPCR x DF_Ex
   Where:
   [miRNA]_{raw value} = the miRNA quantified by commercial dPCR software;
   DF_RT = the dilution factor of the template in reverse transcription;
   DF_dPCR = the dilution factor of the template in dPCR;
   DF_Ex = the dilution factor in the extraction.

6. Synthetic Oligonucleotide Dilution Series

1. Thaw lyophilized synthetic oligonucleotide on ice. Briefly centrifuge it.
2. Dilute the lyophilized synthetic oligonucleotide in nuclease-free water to a final concentration of 10 pmol/µL. Briefly centrifuge it.
3. Dilute it in nuclease-free water as described in Table 5. Centrifuge the dilution briefly between each dilution step at 8,000 x g for 10 s.
4. Continue with the reverse transcription as described in step 2 and analyze the samples with digital PCR as described in step 3 and step 4.

Results

Digital PCR combined with fluorescent hydrolysis probes enables researchers to directly quantify the absolute amounts of specific miRNAs in copies/µL. As the sample in dPCR is partitioned in approximately 20,000 individual PCR reactions, dPCR does not require technical replicates¹⁰. The dPCR system utilizes a mathematical Poisson statistical analysis of fluorescent signals (differing between positive and negative reactions) enabling an absolute quantification witho...

Discussion

Digital PCR is a relatively novel end-point method of PCR that allows the direct absolute quantification of nucleic acids within a sample. The method possesses particular advantages, including a decreased variability, an increased day-to-day reproducibility, and a superior sensitivity^11,12. Further, due to the partitioning of the sample into approximately 20,000 single reactions and endpoint analyses, dPCR is more robust to interfering substances in PCR compared ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RNA-Extraction
miRNeasy Serum/Plasma Kit (50)	Qiagen-Sample & Assay Technologies, Hilden, Deutschland	217184	Kit for microRNA extraction. Kit contains commercial buffer RWT (called number one in the manuscript)  and RPE (called number two in manuscript).
miRNeasy Serum/Plasma Spike-in-Control; Syn-cel-miR-39 miRNA; 10pmol	Qiagen-Sample & Assay Technologies, Hilden, Deutschland	219610	Spike-in for normalisation , Sequence: 5'-UCACCGGGUGUAAAUCAGCUUG-3'
Reverse Transcription
TaqMan MicroRNA Reverse Transcription Kit (1000 Reactions)	Applied Biosystems, Inc., Foster City, CA, USA	4366597	Kit for microRNA reverse transcription
TaqMan MicroRNA Assays M	Applied Biosystems, Inc., Foster City, CA, USA	4440887	Assays used in reverse transcription
hsa-miR-499 (750 RT/750 PCR rxns)	Applied Biosystems, Inc., Foster City, CA, USA	4440887	Assay Number 001352
cel-miR-39 (750 RT/750 PCR rxns)	Applied Biosystems, Inc., Foster City, CA, USA	4440887	Assay Number 000200
PCR Plate, 96-well, segmented, semi-skirted	Thermo Fisher Scientific, Waltham, MA, USA	AB0900	96 well plate for reverse transcription
Microseal ‘B’ seal Seals	Bio-Rad Laboratories, Inc., Hercules, CA, USA	MSB1001	Foil to ensure proper storage
C1000 Touch Thermal Cycler	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1851196	Cycler used for reverse transcription
Droplet Digital PCR
100 nmole RNA oligo hsa-miR-499-5p	Integrated DNA Technologies	Custom	Sequence: 5'-phos-UUAAGACUUGCAGUGAUGUUU-3'
ddPCR Supermix for Probes (No dUTP)	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1863024	Supermix used in droplet generation
TaqMan MicroRNA Assays	Applied Biosystems, Inc., Foster City, CA, USA	4440887	Assays used in digital PCR (fluorescent hydrolysis probe)
hsa-miR-499 (750 RT/750 PCR rxns)	Applied Biosystems, Inc., Foster City, CA, USA	4440887	Assay Number 001352, commercial primers
cel-miR-39 (750 RT/750 PCR rxns)	Applied Biosystems, Inc., Foster City, CA, USA	4440887	Assay Number 000200, commercial primers
DG8 Cartridges and Gaskets	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1864007	Cartridge takes up to 8 samples for droplet generation
DG8 Cartridge Holder	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1863051	Holds cartridges in droplet generation
Droplet Generation Oil for Probes	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1863005	Oil used in droplet generation
ddPCR 96-Well Plates	Bio-Rad Laboratories, Inc., Hercules, CA, USA	12001925	96 well plate for ddPCR
PCR Plate Heat Seal, foil, pierceable	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1814040	Pierceable foil, compatible with droplet reader
ddPCR Droplet Reader Oil	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1863004	Oil used in droplet reading
QX100 or QX200 Droplet Generator	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1863002	Droplet Generator, generates the droplets from sample/oil emulsion
PX1 PCR Plate Sealer	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1814000	Seals the plate before PCR
C1000 Touch Thermal Cycler	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1851196	Cycler used for ddPCR
QX100 or QX200 Droplet Reader	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1863003	Reads PCR-positive and PCR-negative droplets with an optical detector
ddPCR Buffer Control for Probes	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1863052	Blank control and to fill up the remaining wells of 8-well cassette
Software
QuantaSof Software	Bio-Rad Laboratories, Inc., Hercules, CA, USA	1864011	Program for droplet reading
Prism Windows 5	GraphPad Software Inc., La Jolla, CA, USA		Program for statistical analysis