Real-Time Polymerase Chain Reaction MicroRNA Detection Based on Enzymatic Stem-Loop Probes Ligation

Abstract

MiRNAs (microRNAs) are a group of endogenous, small noncoding RNA with the length of 18-25 nucleotides, which have recently been demonstrated to play important roles in a wide range of biological processes. In this work, we developed a simple, sensitive, specific, and inexpensive assay through the combination of enzymatic probe ligation and real-time PCR amplification for the measurement of mature miRNAs. A couple of novel DNA probes with a stem-loop structure were implemented to reduce nonspecific ligation by at least 100-fold. The assay has several remarkable features including wide dynamic range, low total RNA input (0.02-0.2 ng), distinct anti-interference from precursor miRNAs (signal-to-noise ratio > 500), and single-base mismatch discrimination among miRNA sequences. In addition, a one-tube assay could be accomplished by designing a couple of universal probes, which makes it feasible to examine the expression of a whole family of miRNA (such as let-7) at one time. Finally, we validated the method for quantifying the expression of four mature miRNAs including miR-122, miR-1, miR-34a, and let-7a across 10 mouse tissues, where U6 snRNA could be simultaneously examined as an endogenous control. Thus, this method revealed a great potential for miRNA quantitation in ordinary laboratory studies and clinical diagnoses.

Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification

“…A great deal of effort has been made on developing miRNA detection methods, including modified Northern blot (Lee et al 1993;Lagos-Quintana et al 2002), invader assay (Allawi et al 2004), splinted ligation (Maroney et al 2007), and LNA probes-based assay (Weinholds et al 2005;Kloosterman et al 2006). Compared with these aforementioned methods, several real-time RT-PCR-based approaches are becoming a prominent tool for miRNA quantitative analysis due to their high sensitivity and specificity (Chen et al 2005;Raymond et al 2005;Shi and Chiang 2005;Li et al 2009). …”

Multiplexed Detection and Label‐Free Quantitation of MicroRNAs Using Arrays of Silicon Photonic Microring Resonators

“…[15] Although creative approaches that enable reverse transcriptase/PCR amplification have been developed, [16][17][18] many conventional miRNA analyses are prone to sequence-biased amplification or hindered by the need for large amounts of sample. The most widely reported technique for miRNA analysis technique, Northern blotting, requires substantial amounts of starting material, is extremely laborious, and is not amenable to large-scale multiplexing.…”

miRNA Optical Detection

“…However, the expensive thermal cycle instrument limits its expansion application in point-of-care diagnostics. Isothermal amplification techniques free of thermal cycle instrument: including ligase chain reaction (LCR) [40], rolling circle amplification (RCA) isothermal reaction [41], loop-mediated isothermal amplification (LAMP) [42], and EXPAR [43] have emerged as alternative nucleic acids strand amplification techniques in bioanalysis.…”