Analysis of microRNA expression by qPCR

Alteration of microRNA (miRNA) expression in a disease compared to a healthy state and/or correlation of miRNA expression with clinical parameters (like disease progression or therapy response), may indicate that miRNAs can serve as clinically relevant biomarkers_1-3. An important first step for further functional characterisation is the information about differential miRNA expression in cellular processes such as; differentiation_4,5, proliferation or apoptosis₆, that may determine which disease causing genes are specifically regulated by miRNAs, or vice versa; which genes regulate miRNA expression.

Whatever the question you would like to address, the precise information about the level of miRNA expression in a specific cell type or tissue is often considered an important first step. A range of methods can be used for the isolation and profiling of miRNAs. Two recent reviews on microRNA₇ and qPCR₈ in European Pharmaceutical Review addressed both topics individually in great detail, but not their combination. This article aims to provide an insight into the application of quantitative real-time PCR (qPCR) to assay microRNA expression.

miRNA expression profiling using qPCR

miRNA specific qPCR assays are frequently used to validate data obtained from microarray experiments. On first glance, major advantages of this technology over microarrays are; (i) the speed of the assay, (ii) the increased sensitivity with which miRNAs expressed even at low levels can be measured, (iii) the extended dynamic range compared to microarray analysis and (iv) the low requirement for starting material (10 ng/reaction). If, however, the aim is to perform ‘genome-wide’ miRNA profiling of all 722 human miRNAs currently annotated in miRBase (http://microrna.sanger.ac.uk/sequences/index.shtml, release 10; August 2007) by qPCR, together with technical replicates and non-template controls (NTC), six 384 well plates will be required for each condition analysed.

Generally, microRNAs represent challenging molecules to assay. The challenges of miRNA analysis reside in; (i) the small size of the mature miRNAs (18-24 nt), (ii) absence of a common anchor sequence, such as a poly(A) tail or a cap, (iii) highly heterogenous sequence composition of individual miRNAs, resulting in relatively large intervals of melting temperatures (Tm) of nucleic acids duplexes (45-74º C), (iv) in presence of miRNA families within which individual members may differ by just one base (let-7 family, for example). However, each family member may have a different level of its expression, and (v) in their still growing count and changing sequences at some of them.

The absence of a common anchor sequence makes it necessary that for cDNA synthesis, either miRNA specific reverse transcription primer have to be designed for each miRNA analysed, or RNA molecules have to be enzymatically modified (polyadenylated) in order to prime reverse transcription of miRNAs with oligo(dT) primer. Large Tm interval can be narrowed and normalised by use of locked-nucleic acids (LNA).9,10 Currently, several different approaches to determine expression levels of mature miRNAs by qPCR analysis are described in the literature, from which the two discussed below are employed most often.

Detection of miRNA precursors by qPCR

Schmittgen et al.11 have developed a technique for detection of miRNA precursors (pre-miRNA) by qPCR. Based upon an assumption that pre-miRNAs and matured miRNAs are present in one to one ratio, it was thought that this technique could ultimately substitute for the detection of mature miRNAs. However, it is now recognised that miRNAs biogenesis is a complex and tightly regulated process, with the potential of each of its steps being individually regulated, resulting in a ratio between expression levels of pre-miRNA and the mature miRNA different from one another: it is important to note that this ratio may be affected in pathophysiological states12. The underlying experimental principle is similar to the one of standard qPCR analysis of mRNAs with the exception that a primer specific for pre-miRNA is used to prime reverse transcription. Extensive removal of the genomic DNA and the enrichment for the low molecular weight RNA fraction11 is a prerequisite for this method because the amplification primers will also recognise genomic DNA contaminants and miRNA primary transcript (pri-miRNA).

qPCR detection of matured miRNAs via specific reverse transcription primers

Despite the short length of mature miRNAs, specific complementary reverse transcription (RT) primers with an adaptor 5’part can be directly annealed to the miRNA specific sequence to prime the reverse transcription step. The resulting cDNA is then used as a substrate for qPCR reaction with one miRNA specific primer and a second, universal primer whose annealing site is included within the adaptor part of the RT primer. SYBRGreen® incorporated into the amplification products during qPCR enables detection. Approximately 50 ng of total RNA as starting material is required for each qPCR reaction. The often observed disadvantages of this method are; (i) a lack of discrimination between mature, precursor and primary miRNA, and (ii) the absence of a multiplexed reverse transcription step. This system is also available commercially and can be purchased from Applied Biosystems/Ambion (mirVanaTM)9,13.

qPCR detection of mature miRNAs via a synthetic poly(A) tail mediated reverse transcription

In this alternative approach E. coli polyA polymerase (PAP) is employed to synthesise a non-templated homopolymeric polyadenosine tail at the 3’-end of each RNA molecule including miRNAs14. Then, reverse transcription is primed by a primer consisting of two parts: oligo(dT), usually anchored, resides at its 3’part, and a specific adaptor universal primer binding site at its 5’part. The resulting cDNA is then used as a template for qPCR analysis using a miRNA specific primer and the universal primer matching the adaptor sequence of the reverse transcription primer.

qPCR detection of mature miRNAs via reverse transcription with stem-loop primers

A third approach utilises special stem-loop structured primers15 that in their 3’-end part are complementary to and perfectly match a couple (~6) of bases at the 3’-end of a particular miRNA. Due to their structure and sequence, these primers can prime reverse transcription reaction only from mature miRNAs. The resulting cDNAs are amplified during qPCR with a miRNA-specific and a universal primer. In this set up, another level of specificity is implemented by addition to the qPCR mixture of the individual miRNA-specific dual-labelled, hydrolysis TaqMan® probe that is also used for detection of the amplification product. This high specificity is due to hybridisation of the probe to the central region of the amplified PCR product16, in this case to a particular miRNA. The assay utilises the 5’nuclease activity of the DNA Taq polymerase to hydrolyse the hybridisation probe, which carry both a fluorescent reporter and a quencher in adjacent positions, bound to its target amplicon.The emission of fluorescence released upon separation of reporter and its quencher is proportional to the amount of PCR products generated and this will allow accurate quantitation of assayed cDNA. The issue of the miRNA length constraint is dealt with by utilising so-called 3’-minor groove binders (MGB).17 TaqMan® probes must meet certain specifications to function properly. Therefore their design may not be easy due to the miRNA-space constraints and also their optimisation may be more demanding than SYBRGreen® based assays. Also, any change of the cognate sequence of mature miRNA can lead to drop of the corresponding assay’s performance as it was reported recently.18

The qPCR system, marketed by Applied Biosystems (AB), is based upon this principle.

Recently, an application of reverse transcription using stem-loop primer in combination with SYBR®Green detection in the real-time PCR step and thus alleviating necessity for rather expensive TaqMan® probes has been published19.

qPCR detection of mature miRNAs using multiplexing of stem-loop primers for reverse transcription

Genome-wide analysis of miRNA expression using qPCR is both time and reagent consuming. Tang and co-workers20,21 described an approach to reduce both the handling time of the samples and the amount of material required for genome wide analysis of miRNAs through the introduction of a multiplexed reverse transcription step. For example, many miRNAs but not all, contrary to quantification of mature miRNAs via a synthetic poly(A) tail mediated reverse transcription, are reverse transcribed in the same tube. For this purpose they modified the approach described by Chen et al.15 to apply the multiplex RT-PCR principle to the quantification of miRNAs. In a first step, stem-loop RT-primers from their mixture hybridise (anneal) to their corresponding mature miRNAs to enable a multiplexed reverse transcription step. Then, the resulting cDNA is pre-amplified in the presence of low amounts of qPCR primers (pre-PCR; this step is also carried out in a single tube). The pre-PCR amplification product is then diluted and a fraction of the dilution is used for qPCR in 96 or 384 well plates using qPCR primers and TaqMan probes (AB). Applied Biosystems are providing a set of 8 different stem-loop primer pools and recent release of TaqMan assays in their Low Density arrays format can certainly facilitate set up of reverse transcription reactions for miRNAs represented in the particular pool and following qPCR assays on the array.

miRNA qPCR assays also require controls

Current consensus is that for qPCR, normalisation to endogenous control (reference) genes is possibly the appropriate method to correct for variation and efficiency biases. Requirements on these endogenous controls for miRNA qPCR analysis are identical to controls used in mRNA profiling: their gene expression should be stable and in the similar range as targets across as many sample types as possible.

Conclusion

miRNA expression profiling using qPCR is undoubtedly a very powerful approach exploiting all qPCR features: high specificity and sensitivity, wide dynamic range, speed, straightforward set up and scalability. The results are generally robust and reliable although discrimination of expression levels of individual members of miRNA families is not always easy to achieve.

Another point to consider is the sample itself. Regardless of the type of qPCR assay, used to determine miRNAs expression levels, or any miRNAs profiling assay generally, it is critical to realise that some methods applied to purify total RNA, in particular those employing column filtration, can considerably influence the resulting miRNA profile. One should always verify that the sample is not depleted of its miRNAs. It is advisable not to use purification columns for isolation of total RNA whenever analysis of its microRNAs is anticipated, but rather utilise methods based on extraction of RNA by acid phenol in combination with guanidinium-thiocyanate and chloroform22 (nowadays known also as “Tri-reagents” and obtainable from several vendrors under various brand names). Sample source is also important. Generally, formalin-fixed paraffin-embedded (FFPE) specimens are considered a treasure trove of invaluable (particularly clinical) information, but are also difficult for purification of total RNA of acceptable quality suitable for profiling approaches. However, a recent report23 on analysis of samples isolated from fresh, frozen and FFPE specimens describes acceptable correlation between results of miRNA profiling by microarray and qPCR from both specimens’ types. It has been observed that degradation of total RNA does affect expression profiles of miRNAs.

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Dr. Vladimir Benes

Vladimir Benes currently holds a position as molecular biologist, Head of Genomics Core Facility in EMBL Heidelberg, Germany. He has extensive expertise in sequencing, qPCR as well as in microarray profiling for expression and location analyses. Analysis of microRNA expression is the newest addition to the GeneCore activities. He has been heavily involved in providing training courses on various functional genomics techniques.