Stand-Alone Rolling Circle Amplification Combined with Capillary Electrophoresis for Specific Detection of Small RNA

Abstract

Noncoding small RNAs play diverse, important biological roles through gene expression regulation. However, their low expression levels make it difficult to identify new small RNA species and study their functions, calling for the development of detection schemes with higher simplicity, sensitivity, and specificity. Herein, we reported a straightforward assay that combined the stand-alone rolling circle amplification (RCA) with capillary electrophoresis (CE) for specific and sensitive detection of small RNAs in biological samples. In order to enhance the overall reaction efficiency and simplify the procedure, RCA was not preceded with ligation, and a preformed circular probe was employed as the template for the target small RNA-primed isothermal amplification. The long RCA product was digested and analyzed by CE. Two DNA polymerases, the Phi29 and Bst, were compared for their detection performance. Bst is superior in the aspects of specificity, procedure simplicity, and reproducibility, while Phi29 leads to a 5-fold lower detection limit and is able to detect as low as 35 amol of the target small RNA. Coamplification of an internal standard with the target and employment of the RNase A digestion step allow accurate and reproducible quantification of low amounts of small RNA targets spiked into hundreds of nanograms of the plant total RNA extract with a recovery below 110% using either enzyme. Our assay can be adapted to a capillary array system for high-throughput screening of small RNA expression in biological samples. Also, the one-step isothermal process has the potential to conveniently amplify a very limited amount of the RNA samples, e.g., RNA extracted from only a few cells, inside the capillary column or on a microchip.

Expanding Possibilities of Rolling Circle Amplification as a Biosensing Platform

“…This type of RCA is called RNA-primed RCA (RPRCA) and, indeed, RPRCA is used for microRNA and small RNA detection without reverse transcription. 21,40,[42][43][44][45][46][47][48] When several circular ssDNA probes are tested in detecting in vitro transcribed GFP messenger RNA (mRNA) as a standard RPRCA procedure, the RPRCA reaction is observed under a certain ssDNA probe (Fig. 4).…”

Aptameric Molecular Switch for Cascade Signal Amplification

“…Thus, the development of assay signal amplification paths has become important, and several amplification techniques have been developed, including catalytic nanoparticles (1,2 ), PCR (3,4 ), rolling-circle amplification (5,6 ), enzyme-triggered strand displacement amplification (7)(8)(9)(10), entropydriven signal amplification (11,12 ), and cascade signal amplification (13,14 ). The PCR is currently the most widely used thermal-cycling technique for DNA amplification.…”

Recent Progress of On-line Combination of Preconcentration Device with Microchip Electrophoresis

“…In the near future, it is expected that further connection of microfluidic PCR devices such as rolling circle amplification [64,65] with MCE will be developed to realize a high-throughput DNA analysis.…”