MicroRNAs as potential clinical biomarkers: emerging approaches for their detection

Abstract

MicroRNAs (miRNAs) have emerged as novel post-transcriptional regulators of gene expression. These short non-coding RNAs are involved in diverse biological processes and their dysregulation is often observed under diseased conditions. Therefore, miRNAs hold great potential as clinical biomarkers of physiological and pathological states and extensive efforts are underway to develop efficient approaches for their detection. We review recent advances and discuss the promises and pitfalls of emerging methods of miRNA profiling and detection.

Fig. 1

Potential applications of miRNAs as clinical biomarkers. Based on our current knowledge, several clinical applications of miRNAs are proposed as biomarkers of diagnosis, prognosis, therapy response and follow-up, and molecular classification of the disease.

Fig. 2

Invader assay. A) The assay involves two reactions. In the primary reaction, invasive and probe oligonucleotides with hairpin overhangs for enhancing stability are bound to the target miRNA. Hybridization results in the formation of a 5′ overlap-flap structure from the probe oligonucleotide, which serves as a substrate for the structure-specific 5′ nuclease, which causes release of the 5′ flap. In the secondary reaction, a FRET oligonucleotide labeled with a fluorophore (F) and a quencher (Q) is added to a secondary reaction template (SRT). The released 5′ flap from the primary reaction acts as an invasive probe in the secondary reaction, which leads to the formation of a second fluorophore-conjugated overlap-flap structure. Cleavage of this 5′ flap releases the fluorophore to generate a signal that can be quantified. B) An arrestor oligonucleotide complementary to the probe oligonucleotide is used in the secondary reaction that binds to the uncleaved probe to avoid its interference in the binding of the FRET probe to SRT, which reduces the background noise.

Fig. 3

Bioluminescence based assay. A) Quantum dot labeled oligonucleotide probe (QD-probe) and Rluc labeled oligonucleotide probe (Rluc-probe) are used in BRET based detection. In the absence of target nucleic acid, QD-probes hybridize with Rluc-probe to generate a signal after addition of coelenterazine. When the target is present, the BRET signal is decreased owing to the competitive inhibition of Rluc hybridization with QD-probe. B) In a modified assay, biotinylated anti-miRNA probes first are immobilized on neutravidin-coated microtiter plate, then Rluc-labeled target miRNAs and unlabeled synthetic target miRNAs are added. Both sample (Rluc labeled) and synthetic (unlabeled) miRNAs compete with each other during hybridization with the immobilized probes. Upon addition of coelenterazine, a decrease in signal intensity is measured as an estimate of target miRNA levels.

Fig. 4

SPRI based method. Probes partially complementary to the target miRNA are immobilized on a metal based surface (gold/silver SPRI surface). Target miRNA hybridizes with the probes leaving a 3′ overhang. Subsequently, a poly(A) tail is added onto the 3′ overhang and later poly(T) coated gold nanoparticles are adsorbed onto the poly(A) tails and detected with SPRI.

Fig. 5

Electrical detection based assay. A) Sodium periodate derivatives of target miRNAs are hybridized to probes captured on a metallic electrode followed by addition of OsO₂ nanoparticles and incubation to allow their ligation with hybridized miRNAs. Subsequently, electrochemical signals are generated owing to the oxidation of hydrazine. (PD = 1, B) To increase sensitivity, target miRNAs are labeled directly with a electrocatalytic moiety, i.e., Ru(PD)₂Cl₂ 10-phenanthroline-5,6-dione), and hybridized to captured probes. Owing to excellent electrocatalytic activity of Ru(PD)₂Cl₂ toward the oxidation of hydrazine, low concentrations of miRNA can be detected using this method.

Fig. 6

Schematic of the RuO₂-initiated aniline polymerization based biosensor for miRNA detection. PNA probes are assembled on an electrode and target miRNAs tagged with RuO₂ nanoparticles are allowed to hybridize with PNA probes; an aniline/H₂O₂ mixture then is added. Catalytic activity of RuO₂ nanoparticles facilitates polymerization of aniline, which causes deposition of polyaniline (Pan) on the miRNA template and the signal is monitored.