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Explore the power of knock-out cell lines for your research

MicroRNA assay methods: a review of current technologies

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  • Learn about our miRNA assays
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                    At approximately 22 base pairs long, microRNAs (miRNAs) are a comparatively small target to experimentally verify and quantify in the lab.

                    Although bioinformatics plays a big part in identifying putative miRNAs, they also need to be experimentally verified in the lab. A range of techniques has been developed to overcome the challenges of miRNA profiling.

                    Here we review the most popular methods currently in use. Use our table to see at a glance which method is most suited for your experiments and click on a technique for more detail.


                    TechniqueWhen to useBenefitsDrawbacks
                    qPCR
                    • Small scale experiments (1–2 samples or miRNAs)
                    • Established protocols
                    • High sensitivity and specificity
                    • Labor intensive to scale
                    • Requires quality miRNA annotation
                    miRNA arrays
                    • Larger studies (has been used for up to 900 samples)
                    • Established protocols
                    • Purpose built analysis tools
                    • Least quantitative
                    • Requires quality miRNA annotation
                    RNA-seq
                    • Discovery phase research
                    • Whole genome analysis
                    • Single base resolution
                    • Does not require miRNA annotation
                    • Less sensitive than qPCR
                    • Requires most input material
                    • Good degree of technical and bioinformatics skill necessary
                    Multiplex miRNA profiling
                    • Multiplex studies (68 miRNAs, has previously been used for up to 600 samples)
                    • High sensitivity and specificity
                    • Straightforward data analysis
                    • Can be used on crude biofluids
                    • Requires standard lab equipment
                    • Not ideal for small scale experiments of 1–2 samples
                    • Requires quality miRNA annotation

                    miRNA qPCR assays

                    One of the most popular techniques for validating and accurately quantifying miRNAs is quantitative real time PCR (qPCR). As well as being sensitive and quantitative, qPCR is also relatively inexpensive and flexible making it the preferred choice for validating novel miRNAs and for use in relatively small experiments.

                    This technique begins with the conversion of miRNA to cDNA. With the length of a miRNA being comparable to that of a typical DNA primer, cDNA synthesis from miRNAs presents its own challenges. The solution to this is to make the molecule longer, either by incorporating a poly(A) tail or stem-loop structure.

                    Once miRNA has been converted to cDNA it can be assayed using the same approach as a conventional qPCR experiment. Amplification is initiated with an miRNA-specific primer and a stem-loop/poly(A) primer. Either SYBR® Green or a TaqMan® probe is used to detect the amplified product.

                    However, qPCR has its limitations: large experiments can become quite labor intensive to perform. Moreover, unlike conventional qPCR, only one flanking primer can be specific to the miRNA, so care must be taken to ensure only one product is being amplified, especially when using SYBR® Green.

                    The short template length can prove a particularly problematic issue when trying to distinguish miRNAs that may only differ by a handful of bases; melting temperatures can be very low and very similar. The use of novel probes such as locked nucleic acids have been developed that mitigate specificity issues, but sample throughput remains a limitation for large studies (Vester et al., 2004).


                    miRNA arrays

                    Arrays are typically chosen for larger studies covering multiple miRNA targets. While they are the least quantitative of the three miRNA assay methods, conventional DNA oligonucleotide arrays are a relatively inexpensive way to measure hundreds of targets at once.

                    Thousands of probes can be easily spotted on slides, or built up by photolithography, potentially enabling the parallel tracking of all known miRNAs. Arrays are probed by hybridizing fluorescently labeled DNA or RNA samples. The brightness of individual spots can be used to infer relative changes in expression between samples.

                    As with qPCR, distinguishing similar sequences may be problematic, but careful selection of control probes, stringent washing and analysis can mitigate the issue. The maturity of the array platform is a significant benefit here, as there are well developed protocols and purpose built analysis tools available.


                    RNA-seq

                    This method of miRNA quantification uses the high-throughput capability of next-generation sequencing (NGS) platforms. While it cannot quantify miRNA levels with the molar resolution of qPCR, deep sequencing of miRNA does have the advantage of being able to sample all miRNAs present in a sample, whether the researcher knows the sequence or not, making it an ideal discovery tool. Furthermore, as sequences are read directly, RNA-seq can distinguish closely related miRNAs and isoforms.

                    This method has a number of advantages: it can distinguish miRNAs at a single base resolution, it doesn’t require upfront knowledge of an miRNA’s existence, it can determine relative miRNA expression levels and, better yet, tagged libraries can be used for multiplexing.

                    However, of all the methods discussed, NGS does require the most input material, and even then miRNA-seq can’t match the sensitivity of qPCR.

                    Library construction, especially amplification, is potentially a sizeable source of bias and requires a good degree of technical skill to undertake (Baker et al., 2010). Data analysis can also be relatively challenging, especially for a labs that may not have a resident bioinformatician.


                    Multiplex miRNA profiling

                    Multiplex miRNA profiling assays using Firefly particle technology are a more recent addition to the range of tools available to assay miRNAs. A key benefit of this technique is its ability to allow the validation of multiple miRNAs across a range of samples, without the labor intensive workflow or large sample requirement of other techniques.

                    This technique is dependent on hydrogel particles that contain custom selected probes against target miRNAs. miRNAs bind to these probes and are then ligated to adaptor sequences for detection or pre-detection amplification.

                    Particles are optimized for use with common bench-top flow cytometers, allowing detection without specialized lab equipment. In addition, data analysis is relatively straightforward and does not require advanced bioinformatics skills.

                    This technology can be used directly with crude biofluids, and preparation of an RNA library is not necessary. The high sensitivity of the assay means that miRNA profiling can be achieved from input of as little as 10 μl of plasma or serum, or 100 pg purified RNA.

                    Which tool is best?

                    In truth, each of the techniques described can be considered largely complimentary; experimental aims and material considerations will likely dictate which tool a lab opts for first.

                    Studies focused on one or two miRNAs with relatively few test groups are likely to opt for qPCR as the primary assay, whereas experiments trying to discover new miRNA variants will look to an NGS solution. For larger studies examining multiple miRNAs at once, microarrays or multiplex miRNA assays using Firefly particle technology are more suitable.

                    Whatever the choice, given the difficulties involved in handling, verifying and interpreting miRNA data, the consensus view is that best practice is to verify the results of one technique by using a second where possible (Baker et al., 2010).

                    References

                    • Baker M (2010). MicroRNA profiling: separating signal from noise. Nature Methods 7, 687–692.
                    • Vester B, Wengel J (2004). LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43, 13233–13241.


                    Firefly® is a registered trade mark in the Unites States and is registered as a European Union Trade Mark.

                    FireflyTM: a new multiplex platform for miRNA profiling

                    Using the FireflyTM technology we have developed new Multiplex Cellular and Multiplex Circulating miRNA Assays that allow detection of up to 70 miRNAs simultaneously within multiple samples, allowing researchers to be able to conveniently and cost-effectively scale up their experiments.

                    ​​​​​​The platform is built on bio-inert hydrogel particles that give the assays PCR-sensitivity and high-specificity. As a result the assays can profile mutiple miRNAs directly from as little as 10 μl of serum or plasma with no need for RNA purification, or from less than 100 pg​ of purified RNA.

                    ​Our simplified workflow eliminates the need for complicated molecular biology steps typically associated with qPCR and library preparation for sequencing. Morevover the assays can be run on bench-top flow cytometers and come with an associated data analysis software that can be run with no bioinformatics expertise making the implementation of the assay extremely easy.
                    ​


                    Discover our new multiplex miRNA assays >

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