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miRNA assays in the clinical laboratory: workflow, detection technologies and automation aspects

  • Andreas Kappel EMAIL logo and Andreas Keller
Published/Copyright: December 17, 2016
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 55 Issue 5

Abstract

microRNAs (miRNAs) are short non-coding RNA molecules that regulate gene expression in eukaryotes. Their differential abundance is indicative or even causative for a variety of pathological processes including cancer or cardiovascular disorders. Due to their important biological function, miRNAs represent a promising class of novel biomarkers that may be used to diagnose life-threatening diseases, and to monitor disease progression. Further, they may guide treatment selection or dosage of drugs. miRNAs from blood or derived fractions are particularly interesting candidates for routine laboratory applications, as they can be measured in most clinical laboratories already today. This assures a good accessibility of respective tests. Albeit their great potential, miRNA-based diagnostic tests have not made their way yet into the clinical routine, and hence no standardized workflows have been established to measure miRNAs for patients’ benefit. In this review we summarize the detection technologies and workflow options that exist to measure miRNAs, and we describe the advantages and disadvantages of each of these options. Moreover, we also provide a perspective on data analysis aspects that are vital for translation of raw data into actionable diagnostic test results.

Keywords: assay; diagnostics; miRNA; microarray; NGS; PCR

Introduction

Originally discovered in the early 1990s [1], microRNAs (miRNAs) are small (18–24 nt long) RNAs that regulate gene expression mainly at the post-transcriptional level [2], [3]. Following their transcription, a miRNA processing complex consisting of the RNase Drosha and the cofactor DGCR8 cleaves the so-called pri-miRNAs. Thereby, the precursor miRNA (pre-miRNA) is built, which is characterized by a stem-loom structure [4], [5]. The pre-miRNA is rapidly exported from the nucleus into the cytoplasm, and is then cleaved by the enzyme Dicer to produce a short duplex molecule of mature miRNA. Finally, mature miRNAs are loaded into the RNA-induced silencing complex (RISC) where they are bound by a member of the Argonaute (AGO) family of RNA-binding proteins. The RISC complex can then bind to mRNAs that bear sequences which are complementary to the respective miRNA, which can either lead to degradation or to inhibition of translation of the mRNA, respectively [2]. Although miRNAs represent only a small fraction of the total cellular RNA, the copy numbers of mature miRNAs often exceed the copy numbers of their complementary mRNA, thus allowing a tight regulation of the respective complementary mRNA expression [6]. Many if not most miRNA sequences discovered so far are accessible through the database miRBase (http://www.mirbase.org), which is therefore a valuable resource for miRNA expression studies [7], [8]. Besides the miRNAs annotated in miRBase, different studies report evidence for even more miRNAs [9], [10]. Especially in the light of significant bias in high-throughput techniques such as next generation sequencing (NGS), respective novel miRNA candidates however, deserve further experimental validation [11].

Due to their pivotal function as regulators of gene expression, miRNAs play a critical role not only during physiological but also in pathological processes [12]. De-regulated expression of miRNAs will directly impact expression of their target mRNAs, which can therefore be indicative but also causative for a given disease. In fact, de-regulated miRNA expression has been correlated to many human diseases, including various cancers, cardiovascular diseases, neurological disorders, and others [13]. Although examples exist where the deregulated expression of a single miRNA is indicative of a physiological or pathological state, the simultaneous analysis of the expression of multiple miRNA usually provides a better sensitivity and specificity to detect the respective state. Certain miRNAs, e.g. miR-144, have been shown to be dys-regulated in a wide variety of different diseases, including cancer- and non-cancer pathologies [14]. Therefore a large part of candidate miRNA tests aims at measuring panels or signatures consisting of several miRNAs simultaneously rather than single miRNAs alone. Despite substantial advances in miRNA-related disease research, the applicability in clinical research has not finally been demonstrated [15].

It is obvious that the most direct approach to measure a de-regulated miRNA expression would be in the tissue of interest itself, and several test formats that measure miRNA extracted from tissue have been described [16], [17], [18]. However, this is very challenging if not impossible for most clinical questions. For example, a potential screening test for a specific cancer can per definition not be performed on the tissue of that cancer. Or – as it is the case for neurological disorders such as Alzheimer’s disease (AD) or multiple sclerosis (MS), or for cardiovascular disorders such as acute myocardial infarction (AMI) – it is challenging or even impossible to take a tissue sample for analysis of the respective miRNAs. It was therefore an important discovery that miRNAs present in body fluids – for example, urine or saliva [19], but in particular blood and its derivatives such as plasma or serum – can serve as circulating biomarkers [20], [21], [22], [23]. A clear advantage of these sample types is that they can be taken by minimally or even non-invasive procedures, thus providing a clear benefit to the individual to be tested. Moreover, as described above, this approach allows tests to be done in settings where it is difficult or even impossible to obtain tissue samples for miRNA analysis. Finally, some of these sample types – whole blood, plasma and serum – are the standard sample types used in clinical laboratories, thus they are compatible to existing workflows, including sample shipment logistics and sample preparation, respectively. Tests based on these sample types could be offered in most clinical settings, thus providing a good accessibility and availability of these tests to doctors and patients. Hence our review will mainly focus on miRNA tests based on blood and its derivatives.

Sample types and miRNA preparation methods

Blood borne miRNAs are frequently referred to as circulating miRNAs [15]. The remarkable stability of miRNAs in blood, serum or plasma makes respective tests particularly interesting for the clinical routine as sample storage, handling and shipment may not be as critical for miRNAs as for other biomarkers [23], [24], [25], as long as reasonable boundary conditions are taken into account [26]. It is barely understood where the blood-borne miRNA is originating from, but it is plausible that it is derived from multiple sources – certainly from body tissues that show specific miRNA patterns [27], but also from white blood cells, platelets [28], [29], and even microvesicles [30], [31], respectively. A miRNA species that is highly de-regulated in a particular diseased tissue may therefore exhibit only a subtle change in concentration level in blood, as the latter sample type obtains the same miRNA from multiple sources. On the other hand, the level of a blood-borne miRNA species may change in response to a pathological process not only due to changes in expression in the diseased tissue but sometimes may indicate secondary responses, e.g. from white blood cells. This is particularly important when whole blood is used as sample matrix as the extracted miRNA from this sample type contains substantial miRNA from white blood cells.

When using whole blood as sample matrix it is also important to note that in the presence of the standard anticoagulant for this sample type – ethylene diamine tetra acetic acid (EDTA) – miRNA levels in the sample change over time [32], as transcription and degradation of the miRNAs in the white blood cells and platelets continues. Hence blood collection tubes that contain stabilizing reagents that directly lyse the blood cells and thereby immediately stop miRNA expression after the blood draw should be used for isolating miRNA from whole blood. PAXgene™ blood RNA tubes (from PreanalytiX, Hombrechtikon, Switzerland) and Tempus blood RNA tubes (from ThermoFisher, Waltham, MA, USA) are most widely used for that purpose. [26], [33], [34]. Other methods use a chemical extraction method based on concentrated chaotropic salts such as guanidine thiocyanate (e.g. Trizol and QIAzol® reagents) that lyse cells and inhibit RNAse.

For analysis of miRNA expression levels in plasma samples, EDTA [25], heparin [35] or citrate [36] can be used as anticoagulant, respectively. However, as both citrate [37] and heparin [38], [39] have been reported to inhibit Polymerase chain reactions (PCR), EDTA is the anticoagulant of choice when plasma is used for miRNA analysis by quantitative reverse transcription PCR (RT-qPCR).

It is striking that even between serum and plasma there is a difference between miRNA concentration and composition [40], [41], which is due to the release of miRNAs from platelets in the sample tube during coagulation. Notably, miRNAs from plasma and serum differ also in the distribution within the sample types [42]. This also means that miRNA signature are always specific to a given sample type, and a signature that provides a good diagnostic accuracy in plasma may not be valid when the patient sample is serum.

Although methods exist that can measure miRNA without further sample preparation steps [43], most subsequent detection methods require that the miRNAs are purified by methods that are specialized to this substance class [44]. miRNAs are usually purified using a solid-phase extraction procedure on silica columns or beads, or glass-fibre filters, respectively [45]. These purification methods (such as the the miRNeasy® kit from Qiagen, Hilden, Germany, or the miRVana™ PARISTMkit from Applied Biosystems/Thermo Fisher, Waltham, MA, USA) typically have total RNA as output that is enriched in or at least not depleted from miRNAs. This has to be kept in mind, as these “miRNA preparations” indeed contain other RNAs as well. As all these methods differ in their methodology [45], the quality and composition of the output miRNA and thereby the clinical utility of a diagnostic test based on a given miRNA signature will be heavily impacted by the sample preparation method used. This means that a diagnostic test based on a specific miRNA signature is always tied to the sample type and the purification method that was used for its development and validation. One final aspect of the sample preparation step that we would like to mention is that some methods such as the miRNeasy® protocol can be automated, which is important for routine applications in the clinical laboratory.

Detection technologies

After their preparation from respective clinical samples, miRNAs can be analyzed by a variety of technologies [46], [47], [48], [49], [50]. These include electrochemical or potentiometric detection technologies [51], [52], [53], [54], Northern blotting techniques [55], [56], [57], and surface plasmon resonance (SPR) [58], [59]. While the latter methodologies are rather applicable for research application, most (pre)clinical studies dealing with miRNA expression profiling are performed with either of these three technologies: NGS [60], [61], microarrays [62], [63], or RT-qPCR [64], [65], [66], respectively. A summary of respective quantitative techniques and a side-by-side comparison has recently been published as miRQC study [67]. The review articles from Pritchard et al. [46] as well as from de Planell-Saguer et al. [47] provide excellent illustrations of different miRNA detection technologies, which helps to visualize the respective assay concepts. Each of these detection technologies has its advantages and disadvantages for the use in the clinical laboratory, which we would like to summarize in Table 1. In addition to rather established technologies such as RT-qPCR or microarrays, there are also some promising emerging technologies for the measurement of miRNAs in the clinical laboratory environment, which we would like to highlight in a separate chapter and in Table 1.

Table 1:

Comparison of miRNA detection technologies.

Detection methodMajor applicationAdvantagesDisadvantagesReferences
DiscoveryValidationDiagnostic testReady for routine application
qRT-PCR–XXX–Widely available and well-established method–Not suitable for discovery studies[64], [65], [66]
–Excellent sensitivity, specificity and dynamic range
–Ideally for measuring smaller miRNA panels
–Particularly suited for routine measurements in central laboratory settings
NGSXXXX–Broadest spectrum of applications–Not so well suited for high-throughput central laboratory settings[60], [61]
–Very accurate and sensitive
–Can also distinguish variants of a miRNA
–Still very expensive and not everywhere available
MicroarrayXXXX–Established method–Poor degree of automation[62], [63]
–Low throughput, long time to result
–Low sensitivity and specificity
Immunoassay––X––High degree of automation–Sensitivity may not be sufficient for all miRNA panels and sample types[68]
–Particularly suited for routine measurements in central laboratory settings
–Still an experimental method
NanoString–XXX–Very sensitive method with an excellent dynamic range–Very complex workflow[69], [70], [71], [72], [73], [74], [75], [76]
–Method not available everywhere
–High degree of sample multiplexing possible
Electrochemical or potentiometric––X––Particularly well suited for point-of-care tests based on panels of few miRNAs–Technology is not ready for routine applications[51], [52], [53], [54]
–Highly sensitive method
SPR––X––Excellent sensitivity–Low throughput[58], [59]
–Technology is not ready for routine measurements of miRNAs
Northern blotting–X–––Widely available, easy-to-perform method–Laborious and time consuming[55], [56], [57]
–Highly specific
Poor sensitivity

NGS

NGS has recently become a widely accepted approach not only for analysis of gene sequences but also for quantification of expression levels of RNAs [77], [78]. A clear advantage of NGS is that it can quantify known sequences of, e.g. miRNAs while at the same time it can identify and quantify previously unknown sequences. NGS is therefore a valuable approach for both the discovery and the subsequent validation of novel diagnostic or prognostic miRNA signature. Moreover, NGS allows the multiplexed expression analysis of miRNA from different samples in a single experiment, thus eliminating factors that may negatively impact test results. Another clear advantage of NGS is its high dynamic range, which allows to accurately quantifying both highly expressed and low abundant miRNAs at the same time in the same experiment. Finally, NGS allows to detect and quantify variants of a given miRNA species: Mature miRNAs often comprises a distribution of sizes centered around 22 nt, rather than a discrete single length, which is due to 3′ post-transcriptional modifications [79], [80]. It is however, not clear at this stage if the latter aspect is an advantage or a drawback of the methodology, as some of the shortened transcripts being picked up may not represent functional miRNAs [46].

Among the different NGS technologies, the “Sequencing-by-synthesis” technology commercialized by Illumina is now widely used for the discovery of novel miRNA signatures or panels (e.g. [81], [82]), as it provides the depth of sequence data to allow the exact quantification even of rare miRNA species. In this approach, purified miRNAs are first ligated to adaptors at both the 3′ and 5′ ends and subsequently converted into complementary DNA (cDNA). During a subsequent amplification step, index sequences are introduced into the cDNAs that tag the cDNA library and later allow the parallel analysis of several samples in a single experiment. Pooled or single cDNA libraries are then applied to a flow cell where single cDNAs are captured and subsequently bridge-amplified, thus generating clonal spots of each cDNA in the flow cell. The flow cell with the respective spots is then sequenced by a variation of the Sanger method using fluorescence labeled dye terminators, which is followed on-line with a CCD camera. Other NGS technologies such as those based on nanopores have also been evaluated for analysis of miRNA [83] but have still some practical limitations such as a poor accuracy.

Besides for discovery and validation of miRNA signatures, NGS could also be used for measuring rather small miRNA panels from a larger number of samples, as it will be the case for routine testing in the clinical laboratory. However, despite all its advantages, NGS also has several drawbacks: First, NGS is still a pricy methodology, in particular when compared to RT-qPCR. Second, NGS is very time consuming and still is not fully automated, thus tying up resources in the laboratory. Additionally, the data evaluation of NGS is yet not standardized. A recent review that describes only a fraction of available tools lists 129 miRNA tools being used in diverse areas of miRNA research [84], indicating that this space is very fragmented. And finally, although the methodology is increasingly penetrating the research market, it is still questionable if or when the methodology will be routinely applied in the clinical laboratory, which would be the base for a good accessibility of respective tests.

Microarrays

Microarray technology is widely used for gene expression analysis studies and other aspects of genomics [85]. It is based on nucleic acid hybridization between the target molecules that are to be analyzed, and corresponding complementary probes. The strength of microarray-based techniques lies in their ability to quantify large numbers of miRNAs simultaneously in a single experiment, at a prize that is still below the prize for an NGS test. As with NGS, microarrays are therefore the method of choice when it comes to the identification and validation of novel miRNA signature, and microarray therefore currently represents the most widely used high-throughput method for measuring miRNA levels [62], [86], [87]. However, in contrast to NGS, microarrays have a very limited potential for discovery of novel miRNA sequences, nor are they designed to discriminate variants of a given miRNA species from each other, respectively. Only very few studies used microarrays to report novel miRNAs, and in most cases just in a confirmatory manner [88]. It is noteworthy to mention that microarray technology – in contrast to NGS – can also not discriminate between a mature miRNA and its precursor, which may also impact results of an expression analysis study. However, as with NGS, multiplexing (i.e. parallel analysis of different samples such as, e.g. diseased vs. non-diseased samples) is possible when performing microarray experiments. This is a clear strength of the methodology [46].

Microarrays for quantification of miRNAs usually consist of glass slides that carry short oligonucleotides which are either spotted or synthesized in situ. Each spot consists of distinct oligonucleotides that more or less exclusively bind to one single miRNA species. The oligonucleotides can consist of DNA, RNA or other nucleic acids such as LNA (locked nucleic acid). LNA has a higher affinity to miRNA than other nucleic acids, and its use can normalize otherwise highly variable hybridization temperatures of individual sequences [89]. Microarrays can be made in-house, or can be purchased from several commercial suppliers [46]. For example, Agilent (Agilent, Santa Clara, CA, USA) offers microarrays that can measure in parallel all human miRNAs represented in the most recent miRBase version, whereas Exiqon (Exiqon, Vedbaek, Denmark) provides microarrays based on LNA, that can simultaneously measure human, mouse and rat miRNA expression on the same glass slide.

For the actual experiment, purified miRNA is first dephosphorylated at its 3′ end with calf intestinal phosphatase and then labeled with a fluorophore, using T4 RNA ligase. The input amount of total RNA is about 100–150 ng per experiment, which is much less than the amount required for a typical NGS experiment (which is about 1 μg in our experience). This is an important advantage of the microarray technology when sample volume is a critical parameter, e.g. when testing pediatric samples. The miRNA is then hybridized to the microarray for several hours up to days. This long hybridization step, which leads to time to results of sometimes more than 2 days after arrival of a sample in the laboratory, is certainly a major drawback of the technology. After several washing steps that remove any unbound labeled miRNA, the microarray is then scanned for fluorescence, and data are analyzed by bioinformatics tools. While the bioinformatics challenges in microarray analysis are generally lower as compared to NGS, especially the normalization plays a crucial role [90].

The main challenge in applying microarrays or any other hybridization-based technology to measurement of miRNA expression levels arises from the extremely high similarity of miRNA sequences, which sometimes differ only in a single nucleotide yet exhibit completely different expression profiles [91]. This would result in a “cross-talk” of individual hybridization events, meaning that individual oligonucleotide spots of the microarray measure the expression of a panel of different unrelated miRNAs rather than of a single and distinct miRNA. This issue is partially addressed by the use of LNA capture oligonucleotides and the application of rather high hybridization temperatures [92], but it cannot be fully solved. It could therefore be expected that results differ between microarray experiments and other miRNA expression profiling platforms. This effect and others may account for the poor inter-platform reproducibility of miRNA expression results that seems to be a phenomenon that may be addressed by miRNA-specific normalization methods [93]. In line with this observation, we have recently described a noteworthy difference in expression data obtained by microarray or NGS experiments, on a miRNA signature that could differentiate between multiple sclerosis patients and control subjects, respectively [81]. In a discovery approach, we detected 38 significantly deregulated miRNAs by NGS and eight significantly deregulated miRNAs by microarray analysis. Only three individual miRNAs were identified as significantly deregulated by both NGS and microarray analysis, whereas an additional five miRNAs showed the same direction of regulation in the microarray and NGS analysis, i.e. each of these miRNAs was either up- or downregulated in both approaches, although the deregulation of these five miRNAs in the microarray experiments was not statistically significant. As with specificity, also the sensitivity of microarray-based miRNA tests is lower when compared to respective NGS or RT-qPCR tests. This is particularly challenging when miRNAs from body fluids with low miRNA concentrations such as serum are analyzed by standard microarrays, although this is in principle possible (e.g. [94]).

To summarize, microarrays have their clear strength when it comes to discovery and validation of miRNA signatures. However, for routine clinical application, other technologies such as RT-qPCR may be more appropriate. In contrast to NGS, microarrays cannot be used to discover novel miRNAs, and they can also not discriminate between variants of distinct miRNAs.

RT-qPCR

The most straightforward approach to measure the expression level of miRNAs in the clinical laboratory context is certainly by using RT-qPCR [64], [65], [66]. One major reason for this is simply the availability of respective instruments in most laboratories, as RT-qPCR based tests are already routinely used for other in vitro diagnostic applications such as viral load testing or mRNA expression profiling, respectively [95]. The methodology is based on reverse transcription of RNA to cDNA, followed by a quantitative polymerase chain reaction (RT-qPCR). The accumulation of the reaction product is followed in real time; hence the technology is sometimes referred to as “real time PCR”.

Due to the small size of miRNAs, the reverse transcription step for miRNAs is somehow different from the protocols employed for reverse transcription of e.g. viral RNA or mRNAs. Two alternative approaches exist for this step: The first one extends the miRNA at its 3′ end with a poly(a) tail using enzymatic treatment of the miRNA sample with poly(A) polymerase [66], [96]. The resulting polyadenylated RNA, which contains precursor miRNA, other small RNAs, and even mRNA on top of mature miRNA, respectively, is then reverse transcribed using a universal oligo(dT) primer. Qiagen (Hilden, Germany) offers a unique buffer system contained in the miScript II RT Kit, which allows the selective reverse transcription of either miRNA or mRNA. The resulting cDNA is subsequently amplified using a forward primer that is specific to individual miRNAs, and a universal reverse primer such as the miScript Universal Primer. PCR product accumulation is then detected by intercalation with SYBR® Green and fluorescence measurement [97]. As with microarrays, the specificity of the amplification reaction can be further improved by using LNA instead of DNA for the forward primer [98], [99]. A discrimination of precursor and mature miRNA can be achieved by selection of appropriate primers [100].

The second approach for the reverse transcription and subsequent PCR amplification of miRNA is based on a stem-loop primer that is specific to a complementary miRNA [65]. The primer hybridizes to the 3′ portion of the miRNA while at the same time extends the 3′ end of the miRNA with its 5′ end to generate a nicked RNA hybrid consisting of a specific miRNA and a specific stem-loop primer [101]. After reverse transcription, the resulting cDNA is amplified by PCR using a forward primer that is specific to the miRNA sequence, and a reverse primer that is specific to the stem-loop primer. A TaqMan probe that binds to the resulting amplicon is contained within the PCR reaction, and as the DNA polymerase proceeds along template, the TaqMan probe is hydrolyzed so the quencher is freed from fluorescent dye, resulting in light emission.

In order to quantify the expression of miRNAs by RT-qPCR, the Ct (cycle threshold) values of each miRNA measured must be related to a standard. One solution is to spike in non-human synthetic miRNAs to a sample and set the expression levels of the miRNAs of interest in relation to this control [23], [24], [35]. A second approach relates endogenous controls to the expression of miRNAs of interest, such as small RNAs [81]. Finally, it is also possible to use endogenous miRNAs as internal standard, either by using single miRNAs or a small reference panel [102], [103], [104]. Schwarzenbach and co-workers have recently addressed the general challenge of miRNA normalization using small RNAs [105]. In another study, different endogenous controls have been tested on lung cancer and control samples, highlighting that different endogenous controls can indeed lead to varying diagnostic results [106].

As miRNA tests usually measure panels of miRNAs that can consist of dozens of panel members [13], respective RT-qPCR reactions are carried out in a highly parallel approach. For this purpose, commercial kits exist (e.g. the miScript miRNA PCR Arrays from Qiagen, Hilden, Germany, or the TaqMan® MicroRNA Array from Applied Biosystems/Thermo Fisher, Waltham, MA, USA, respectively) that use microwell plates loaded with pre-plated PCR primers, which are more or less ready-to-use for the PCR reaction.

As they can be performed on standard equipment, miRNA tests based on RT-qPCR are widely available, can be automated to a high degree, and are compatible with the workflow in the clinical laboratory, respectively. However, these advantages can only be leveraged once a miRNA test has been established, since for discovery and validation of novel miRNA signatures the NGS or microarray technologies are better suited. Mestdagh et al. have recently reported a systematic comparison of different commercially available platforms for miRNA expression profiling, including microarray, NGS and RT-qPCR [67]. Among the analytical parameters tested, the RT-qPCR tests exhibited the highest sensitivity and showed the most balanced profile of sensitivity, specificity, accuracy and reproducibility, respectively. Moreover, the LNA-based RT-qPCR method from Exiqon exhibited the least cross-reactivity of all methods tested when critical miRNA panels such as the let7 family [91] where analyzed. All these aspects make RT-qPCR an attractive technology for routine testing of miRNAs.

Emerging technologies

Any technology for measurement of miRNAs in the clinical laboratory must meet not only certain analytical performance criteria such as sensitivity and reproducibility, but it should ideally also have a high degree of automation and should use existing workflow elements, respectively.

The NanoString® nCounter Platform (from Nanostring Technologies, Seattle, WA, USA) is one of those promising emerging technologies for profiling of RNA. It is a hybridization-based, medium- throughput technology for measuring mRNA and miRNA levels, which can detect specific nucleic acid molecules from low amounts of starting material without the need for reverse transcription or cDNA amplification [69]. The technology is based on a novel method of direct molecular barcoding and digital detection of target molecules through the use of color-coded probe pairs. For the analysis of miRNA expression levels, a miRNA sample is first annealed with a library of sequence-specific tags to the 3′ ends of the target miRNAs, followed by a ligation reaction and an enzymatic purification step to remove unligated tags. Sequence specificity between a miRNA and its synthetic sequence tag is ensured by a stepwise control of hybridization and ligation temperatures. Control RNAs are normally included in the reaction, which allows for the monitoring of ligation efficiency and specificity throughout each step of the reaction.

After miRNA sample tagging, target-specific probe pairs are hybridized to the sample in solution: The reporter probes carry a fluorescent signal, whereas the capture probes immobilize the resulting complexes on the surface of the cartridge for the subsequent data collection. In the current configuration, 800 pairs of probes specific for a set of miRNAs can be used in parallel. After hybridization of the probe pairs, probe/target complexes are aligned and immobilized in the nCounter Cartridge. Cartridges are then placed in the nCounter Digital Analyzer for data collection. Each miRNA of interest is identified by the “color code” generated by six ordered fluorescent spots present on each Reporter Probe. The Reporter Probes on the surface of the cartridge are then counted and tabulated for each miRNA species.

nCounter miRNA panels have been utilized to differentiate between normal and disease states in cancer [70], neurodegeneration [71], and bowel syndrome [72]. Moreover, respective panels have been successfully used for stratification of patients [73], [74], [75].

When compared to RT-qPCR or in particular microarray-based technologies, NanoString technology has several benefits [76]: First, it is faster than those methods, in particular when compared to microarray. Second, the hybridization method used avoids amplification steps even for low-abundance transcripts, which reduces bias. Third, the measurement principle provides absolute quantification, which also reduces potential bias. Finally, the technology has a higher degree of parallelization than most current PCR tests, which is an advantage for both discovery/validation studies as well as for tests based on more complex miRNA signatures. Moreover, in comparison to microarrays the technology is more sensitive and provides also a better dynamic range, which allows a better detection of up- or downregulation of miRNA expression. However, a major disadvantage of the technology is that it is currently only available in specialized research laboratories and not in routine clinical laboratories. This prevents the accessibility and availability of respective miRNA tests.

In contrast to NanoString platforms, immunoassay analyzers are standard equipment in most routine clinical laboratories. These analyzers are not only widely available, but they are particularly well suited to perform highly automated tests that require almost no manual step from sample arrival at the laboratory to result. The better availability of immunoassays when compared to RT-qPCR or other methods is also reflected by the fact that worldwide immunoassay sales in 2014 made up a total of 14.95 billion US$ (non-isotopic tests only), whereas molecular tests including those for microbiology and blood banking where only 4.48 billion US$ [107]. We thus have recently developed and evaluated a technical option to perform miRNA tests on standard immunoassay analyzers [68], [108]. Our assay measures miRNAs by hybridization of a miRNA sample to a biotinylated DNA oligonucleotide that is complementary to a specific target miRNA, followed by detection of the heteroduplex by a monoclonal antibody [109]. Resulting immunocomplexes can be quantified on standard immunoassay analyzers such as the Advia Centaur® Immunoassay System (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) [110] using e.g. acridinium ester labeling of the antibody for detection [111].

Our prototype assay exhibited a time to result of less than 3 h including sample preparation, and could reliably profile miRNAs at concentrations as low as 1 pmol/L [68]. Moreover, the assay exhibited an analytical specificity of 99.4%, as assessed with highly related miRNA species from the let7 family. Although this assay concept is highly attractive from a workflow point of view, it needs further optimization to meet the requirements of routine laboratory tests, in particular regarding the limit of detection to cover also low abundant miRNAs, as well as measurement of multiple miRNAs per sample (multiplexing), which is not addressed by standard immunoassay workflows today.

Electrochemical detection technologies provide a base for cost-effective, highly sensitive miRNA assays, particularly suited for point of care settings. The various experimental setups that have been evaluated for this detection technology class have been nicely reviewed elsewhere [112], [113]. It is noteworthy to mention that the sensitivity of these methods allows in principle even the quantification of low-abundant miRNAs from serum without pre-amplification [114], [115]. However, none of these electrochemical methods have been validated for their practical utility under routine laboratory conditions so far. This leaves the question open on how respective tests are influenced by sample variability as well as human or external factors, respectively.

Recently, a plethora of novel assay concepts for measurement of miRNAs has been published, including formats based on multiplexed Förster or flourescence resonance energy transfer (FRET) and quantum dot assay designs [116], [117], [118], and ligation-mediated hybridization to microarrays [119], respectively. It remains to be elucidated if any of these methods can take the next step to move into the clinical routine.

Post-analytical aspects: data analysis and result reporting

Especially for applications where timely results are required [120], [121], bioinformatics and result reporting should not become the bottleneck. Generally, computational approaches are the more important the larger the data quantity gets. Especially for NGS, yielding data set size in the range of several GB, fast and accurate data analysis is a challenge. A recent review describes as much as 129 different bioinformatics tools [84]. Even restricting the consideration to approaches for quantifying miRNAs, a manifold of methods has been published. Among the most commonly used tools, miRDeep is applied not only to quantify miRNAs but also to predict potentially novel markers [122], [123]. Similar functionality is offered by the UEA workbench [124], the sRNAtoolbox [125], miRExpress [126] or miRTools [127]. Another program tailored for the quantification of miRNAs from NGS data is miRNAKey [128]. Since the results of respective tools rely on different analytical strategies, starting with different mapping and alignment techniques such as bowtie [129] or Burrows-Wheeler Aligner (BWA) [130] the results between different tools vary. Comparative analyses using NGS data from AD patients [131] highlighted substantial variations in differentially expressed miRNAs dependent on the used tools (all tools were applied with standard parameters, data not shown). In addition, most of the aforementioned approaches have no built-in ability to perform statistical tests to discover differentially regulated miRNAs or to carry out other analyses such as the detection of iso-forms. Here, further tools that report dys-regulated miRNAs such as miFRame [132] are required.

To report differential expression of miRNAs poses additional hitches. One example are different normalization strategies. While all experimental approaches have challenges with respect to data normalization [90], [105], several studies on patient samples describe approaches how to overcome respective normalization issues in body fluids [106] and tissues [133].

Finally, the complex miRNA profiles measured for diseases have to be processed in a manner that clinical experts can interpret the findings.

Conclusions

Although miRNA-based diagnostic tests have not made their way yet into the clinical routine, several promising prototype assays exist that may eventually lead to routine applications. A future routine test based on miRNAs will consist of the following elements, each intimately linked to this respective test: i) a defined sample type, ii) a defined sample preparation method, iii) a detection technology, iv) a data analysis tool, and of course v) a panel of miRNAs that is specifically analyzed in the scope of the test. It is important to understand that these elements are fixed for each test, as the change of any of these elements could significantly impact the overall test results. This is also to be kept in mind when taking a test from its discovery and prototyping phases towards design verification and validation, as any design change may impact results.

Among the detection technologies, NGS, microarrays, and RT-qPCR are currently most widely used, respectively. All these have their advantages and disadvantages, but RT-qPCR is certainly the method of choice for a routine test. It remains to be elucidated if emerging test formats such as the Nanostring technology will play a role in the future. Like with other detection technologies in the laboratory, a solid standardization is needed in order to make test results comparable. Finally, data analysis is key to each and every miRNA test, and selection of the right bioinformatics tools will significantly impact the quality of the test.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Received: 2016-5-31
Accepted: 2016-11-1
Published Online: 2016-12-17
Published in Print: 2017-5-1

©2017 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Not all good things come in big packages
  4. Pre-analytical and Analytical Issues of miRNA Measurement
  5. Variability in, variability out: best practice recommendations to standardize pre-analytical variables in the detection of circulating and tissue microRNAs
  6. Pitfalls of analysis of circulating miRNA: role of hematocrit
  7. Intraindividual variation of microRNA expression levels in plasma and peripheral blood mononuclear cells and the associations of these levels with the pathogenesis of autoimmune thyroid diseases
  8. miRNA assays in the clinical laboratory: workflow, detection technologies and automation aspects
  9. Clinical Applications of the Different Circulating Forms of miRNAs
  10. The role of extracellular vesicle microRNAs in cancer biology
  11. The clinical significance of platelet microparticle-associated microRNAs
  12. microRNAs in lipoprotein and lipid metabolism: from biological function to clinical application
  13. microRNAs in cardiovascular disease – clinical application
  14. miRNAs in Cancer
  15. Non-coding RNAs: the cancer genome dark matter that matters!
  16. miRNAs as novel biomarkers in the management of prostate cancer
  17. Upregulated miR-16 expression is an independent indicator of relapse and poor overall survival of colorectal adenocarcinoma patients
  18. Identification of a novel microRNA, miR-4449, as a potential blood based marker in multiple myeloma
  19. miRNA analysis in pancreatic cancer: the Dartmouth experience
  20. miRNAs and Genomic
  21. miRNAs, single nucleotide polymorphisms (SNPs) and age-related macular degeneration (AMD)
https://doi.org/10.1515/cclm-2016-0467
Keywords for this article
assay; diagnostics; miRNA; microarray; NGS; PCR

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Not all good things come in big packages
  4. Pre-analytical and Analytical Issues of miRNA Measurement
  5. Variability in, variability out: best practice recommendations to standardize pre-analytical variables in the detection of circulating and tissue microRNAs
  6. Pitfalls of analysis of circulating miRNA: role of hematocrit
  7. Intraindividual variation of microRNA expression levels in plasma and peripheral blood mononuclear cells and the associations of these levels with the pathogenesis of autoimmune thyroid diseases
  8. miRNA assays in the clinical laboratory: workflow, detection technologies and automation aspects
  9. Clinical Applications of the Different Circulating Forms of miRNAs
  10. The role of extracellular vesicle microRNAs in cancer biology
  11. The clinical significance of platelet microparticle-associated microRNAs
  12. microRNAs in lipoprotein and lipid metabolism: from biological function to clinical application
  13. microRNAs in cardiovascular disease – clinical application
  14. miRNAs in Cancer
  15. Non-coding RNAs: the cancer genome dark matter that matters!
  16. miRNAs as novel biomarkers in the management of prostate cancer
  17. Upregulated miR-16 expression is an independent indicator of relapse and poor overall survival of colorectal adenocarcinoma patients
  18. Identification of a novel microRNA, miR-4449, as a potential blood based marker in multiple myeloma
  19. miRNA analysis in pancreatic cancer: the Dartmouth experience
  20. miRNAs and Genomic
  21. miRNAs, single nucleotide polymorphisms (SNPs) and age-related macular degeneration (AMD)
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