June 15, 2015 (Vol. 35, No. 12)

Michael Tackett Director of Assays Services Abcam
Graeme Doran Director of Genomic Assays Abcam
Daniel Pregibon General Manager Abcam

Particle-Based Approach Profiles Up to 68 miRNAs from Plasma, Serum, and Exosomes

MicroRNA (miRNA) profiling has tremendous potential for the diagnosis and prognosis of a broad range of diseases including cancer, cardiovascular disorders, and Alzheimer’s disease. Due to their stability in blood, miRNAs are ideal biomarkers; however, the full promise of miRNA profiling has yet to be realized, largely due to the limited throughput of existing validation technologies, lack of compatibility with samples containing PCR inhibitors, and errors introduced during sample preparation.

While there are several multiplexing technologies for miRNA detection, for either in-depth analysis of a few samples (e.g., sequencing) or analysis of a few targets over many samples (typically accomplished using manual qRT-PCR), no platforms currently exist that are ideally suited for the detection of numerous miRNAs over a large number of samples, as is required for the validation of clinical miRNA biomarkers. In addition, the implementation of clinical biomarker assays requires robust methods for sample preparation, typically consisting of specimen collection, RNA purification, and library preparation. 

Another barrier is sample preparation, as methods currently used are not only prone to human error but can have profound effects on RNA yield, sample throughput, and miRNA profile. Furthermore, the presence of PCR inhibitors, such as heparin, may interfere with reverse transcription, rendering many of the qPCR or sequencing approaches unusable. Ideally, miRNAs would be detected directly from clinical samples with no RNA purification using an approach that minimizes or eliminates the effects of PCR inhibitors.


Multiplex Circulating miRNA Assay

The Multiplex Circulating miRNA Assay using Abcam’s FireflyTM Technology was developed to address the needs for miRNA biomarker validation, enabling direct detection of up to 68 miRNAs of choice with PCR sensitivity from crude biofluids including serum, plasma, and exosomes. The platform uses barcoded hydrogel particles to provide researchers with a miRNA profile in every well.

The workflow for the Multiplex Circulating miRNA Assay is simple, consisting of capturing, labeling, amplifying, and recapturing small RNAs for highly sensitive and robust detection (Figure 1). After capture, labeling and amplification of target miRNAs, assay readout is performed using a standard flow cytometer. Data files from the cytometers are interpreted with the Firefly Analysis Workbench software for analysis and export.


Figure 1. Multiplex circulating miRNA assay workflow.

Eliminating Need for RNA Purification

Robust miRNA profiles can be obtained with the Multiplex Circulating miRNA Assay directly from crude biofluids such as serum, plasma, and exosomes with no need for RNA purification. This approach dramatically simplifies the workflow, eliminates an unreliable step in sample preparation, and reduces the absolute amount of starting material required.

To assess the ability of the assay to profile from crude sample, we compared miRNA expression profiles obtained from purified RNA with those obtained from crude serum (Figure 2). For purified RNA samples, TRIzol® LS was used with the recommended protocol starting from an input amount of 250 μL of serum. For the crude samples, 40 μL of serum was used in the digest. 

In both cases, an equivalent of 12.5 μL serum was used as input to the Multiplex Circulating miRNA Assay. There was found to be a close correlation between data from crude serum and purified RNA (Figure 2). The elimination of RNA purification from the miRNA profiling workflow makes the Multiplex Circulating miRNA Assay particularly suited to high-throughput applications where reduction of pre-analytical variability is crucial.


Figure 2. miRNA profiling in crude digests vs. extracted RNA, with Pearson Correlation of 0.97.

Profiling miRNAs in the Presence of Heparin

The presence of PCR inhibitors, such as heparin, in sample collection tubes poses a tremendous challenge for RT-PCR based profiling methods. However, the post-hybridization labeling method used by the Multiplex Circulating miRNA Assay performs well with crude digests of plasma harvested across a variety of collection tubes, including those containing heparin. We assayed blood plasma donated from five patients, collected in four tubes during a single draw. Anti-coagulants included sodium citrate (NaCit), potassium EDTA (K2EDTA), sodium heparin (NaHep), and lithium heparin (LiHep). Each sample was digested and tested in triplicate. Results are shown in Figure 3.

Results were virtually identical across plasma collection methods, with each of four targets spanning a range of expression levels detected with consistent signal across all five patients and four collection types. This ability to profile miRNAs from samples prepared in heparin may provide clinical researchers the opportunity to study sample banks that would otherwise be inaccessible with other profiling approaches.


Figure 3. Profiling miRNA in heparin samples. Average signal was seen across five patients, with error bars representing one standard deviation in the measurements.

Conclusion

The Multiplex Circulating miRNA Assay provides life science researchers with a robust method to profile miRNAs in crude biofluids. The assay is sensitive, specific, and enables high-throughput analysis required for biomarker validation studies. The use of crude biofluid digests eliminates a major source of pre-analytical variability while minimizing workflow. In addition, the Multiplex Circulating miRNA Assay is compatible with a range of plasma collection tubes, including those containing heparin. This assay will provide researchers with a flexible and reliable method for miRNA profiling across a broad range of applications.



























Michael Tackett is director, assays services, Graeme Doran is director, genomic assays, and Daniel Pregibon (dpregibon@abcam.com) is GM, platform innovation at Abcam.

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