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Once extracted from a sample, miRNAs can be quantified in various ways. The most popular methods are microarray analysis, RNA-seq, real time qPCR, northern blotting, and in situ hybridization. No matter which method is used, it is important to always verify the results of one technique by using a second whenever possible (1).

See below for a comparison of some of the most common miRNA detection techniques. Each method has advantages and disadvantages, and will provide a different type of information.

miRNA microarrays are a popular method for performing large scale studies of hundreds of miRNAs at once (2). Here is how it works:

• Acquire two or more samples to be compared. Purify then isolate the miRNA by size.

• A miRNA microarray displays the probes for thousands of identified or theorized miRNAs, which may be pulled from a database such as miRBase (3). Each probe is displayed in a different location on the array called a “pinhole”.

• Ligate fluorescent labels to miRNA then add the sample to the pinholes of the microarray. miRNAs that have complementarity to a given probe will bind and fluoresce.

• A machine measures the level of brightness from each pinhole. Fluorescence levels between different probes and samples are compared. A higher level of brightness indicates a greater quantity of that miRNA.

It is not possible to accurately ascribe a level of brightness to a particular concentration of miRNA, so microarrays are not quantitative. They are best for comparing relative changes in miRNA expression between different samples. One disadvantage of using miRNA microarrays is that distinguishing the presence of similar sequences may be difficult. This is because imperfect binding of a probe to a miRNA may be strong enough to keep a miRNA in place if the chip is not washed enough. Another disadvantage is that it is only possible to probe for miRNAs with known or theorized sequences, so this method cannot be used to discover new miRNAs.

Next Generation Sequencing (NGS) technology allows for the screening of all miRNAs from a single sample, regardless of whether their sequence is known or not. The general steps involved in RNA-seq are:

• Purify total RNA from a sample.

• Optionally, isolate miRNAs by size selection.

• Ligate 5’ and 3’ adapters to the RNA.

• Reverse transcribe the RNA into cDNA then amplify using PCR.

• Purify and sequence the library.

• Analyse sequencing data. This may be done using tools such as miRanalyzer (4), mirTools (5), and the deep-sequencing small RNA analysis pipeline (DSAP) (6).

Real time quantitative PCR (qPCR) is a commonly used method to validate miRNA expression. Due to their small size, traditional qPCR won’t work on mature miRNAs. Therefore, before reverse transcription and amplification are carried out the length of the miRNA must be extended. There are two methods to achieve this: the poly(A) method and the stem loop method.

The first method adds a common poly(A) tail to the 3’ end of all miRNAs, then uses a universal primer to reverse transcribe the miRNA into first-strand cDNA. cDNA is amplified using a miRNA-specific forward primer and a poly(A) reverse primer. Quantification is measured using an intercalating fluorescent dye, such as BrightGreen or SYBR Green.

The second method uses a stem loop primer specific for the miRNA of interest to reverse transcribe it into first-strand cDNA. Once cDNA is present, amplification can be achieved using a miRNA-specific forward primer and a stem loop reverse primer. Quantification is measured using a TaqMan fluorescent probe.

In comparison to other techniques, miRNA real time qPCR has the advantage of being quantitative and highly sensitive. However, only a few miRNAs can be quantified at a time, so large scale studies using qPCR can be laborious. As well, the short template length of miRNA means that it can be difficult to distinguish miRNAs that differ by only one or two nucleotides. New technologies such as locked nucleic acids (LNA) have been developed to improve specificity and differentiation of mature miRNAs from precursors (8).

For more information about RT-qPCR, check out our RT-qPCR Knowledge Base Article.

One of the older methods of miRNA detection, Northern blotting is still commonly used due to its simplicity and low cost. It involves the separation of total RNA on a polyacrylamide gel. The separated RNA is then transferred to a nitrocellulose membrane and detected using a miRNA-specific labelled probe. The probe used may be either a regular DNA oligonucleotide, or a LNA probe for greater sensitivity and specificity.

in situ hybridization is used to visualize miRNA expression within a cell or tissue (9).

Here is a general overview of in situ hybridization for detection of miRNAs.

• Prepare slides with cells or tissue.

• Treat sample to fix RNA in place and increase access of the probe.

• Add a labelled probe, which will hybridize to the target miRNA. Probes may be labelled radioactively, fluorescently, or chromogenically.

• Wash slides.

• Visualize sample.

There may also be a sequence amplification step which allows for the detection of low-quantity miRNAs. Sequence amplification may be done via a Rolling Circle Amplification (RCA) when special probes are used (padlock probes or seal probes). Alternatively, branched DNA techniques can be used, which rely on the binding of multiple labelled secondary probes to each specific primary probe (10).

There are many other technologies being explored to meet the unique challenges of miRNA detection and quantification (11). Recent efforts have focused on the use of nanomaterials to detect miRNAs. Nanoparticles which have been applied to miRNA detection include magnetic particles, quantum dots, gold nanoparticles, silver nanoclusters, and graphene oxide. The use of nanosensors may be combined with other techniques such as SPR, microarrays, and electrical methods. Another new technique relies on capillary-electrophoresis. This method differentiates miRNAs based on their hybridization to probes carrying different drag-tags. The drag-tags will cause the labelled miRNAs to migrate at different speeds during electrophoresis.