Bringing MicroRNAs to Light: Methods for MicroRNA Quantification and Visualization in Live Cells

Abstract

MiRNAs are small non-coding RNAs that interact with their target mRNAs for posttranscriptional gene regulation. Finely controlled miRNA biogenesis, target recognition and degradation indicate that maintaining miRNA homeostasis is essential for regulating cell proliferation, growth, differentiation and apoptosis. Increasingly, miRNAs have been recognized as a potential biomarker for disease diagnosis. MiRNAs can be found in blood, plasma, and tissues, and miRNA expression and activity differ in developmental stages, tissues and in response to external stimuli. MiRNA transcripts are matured from pri-miRNA over pre-miRNA to mature miRNA, a process that includes multiple steps and enzymes. Many tools are available to identify and quantify specific miRNAs, ranging from measuring total miRNA, specific miRNA activity, miRNA arrays and miRNA localization. The various miRNA assays differ in accuracy, cost, efficiency and convenience of monitoring miRNA dynamics. To acknowledge the significance and increasing research interest in miRNAs, we summarize the traditional as well as novel methods of miRNA quantification with strengths and limitations of various techniques in biochemical and medical research.

Keywords: RT-qPCR; fluorescence; microRNA; next generation sequencing; reporter activity.

Figure 1

MiRNA Microarrays. cDNA is synthesized by reverse transcription of extracted miRNAs and the cDNAs are subsequently labeled with e.g., a fluorophore tag. Oligonucleotide probes complementary to target miRNAs are spotted on a carrier plate and the fluorescently labeled cDNA is hybridized. The signal intensity corresponds to miRNA abundance.

Figure 2

RT-PCR based quantification of miRNA. A Stem-loop RT primer binds to the target miRNA either (A) directly or (B) indirectly after addition of adenine nucleotides to the 3′- end, allowing for reverse transcription. The generated cDNA is amplified by PCR with a miRNA specific forward primer and a universal reverse primer. Instead of SYBR green, a gene specific (C) or universal (D) Taqman probe can be used.

Figure 3

Invader assay. Binding of a primary probe and an over lapping invader oligonucleotide to the target miRNA initiates a cleavage reaction carried out by structure-specific 5′ flap nuclease to release the 5′ flap of the primary oligonucleotide probe. The free 5′ flap acts as an invader oligonucleotide for a second reaction to bind to a FRET cassette containing fluorescent (F) and quencher (Q) molecules. When the 2nd structure is recognized, the FRET cassette is cleaved, emitting detectable fluorescence signals.

Figure 4

Luciferase reporter assay. A luciferase reporter gene is fused to the promoter sequence of a gene of interest, e.g., the promoter for a specific miRNA, or a target mRNA. Luciferase is transcribed when the native promoter is active, and activity of the reporter protein is detected by converting luciferin substrate to a detectable bioluminescence.

Figure 5

Green fluorescence protein reporter assay. A green fluorescence protein (GFP) reporter gene is fused to a 3′-UTR encoding miRNA binding sites. GFP mRNA translation is controlled by, and responsive to, changes in miRNA activity. Binding of the miRNA to the 3′-UTR of the reporter construct decreases the production of GFP and detectable fluorescence.