Rolling‐Circle Amplification: Unshared Advantages in miRNA Detection

Neubacher, Saskia; Arenz, Christoph

doi:10.1002/cbic.200900116

Cited by 32 publications

(16 citation statements)

References 20 publications

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“…Previous studies have shown that target-primed RCA is well-suited to the small size of miRNAs (2,15,16). However, the earlier studies required a separate ligase enzyme to carry out the cyclization reaction.…”

Section: Discussionmentioning

confidence: 99%

“…Mature miRNAs are only 19–23 nt in length, often differ from one another by a single nucleotide, and are highly variable in their expression levels in cells (1). These parameters put significant demands on standard RNA detection techniques such as reverse transcriptase–PCR, and require a higher level of sensitivity than a simple fluorogenic reaction can provide (2). …”

Section: Introductionmentioning

confidence: 99%

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Amplified microRNA detection by templated chemistry

Harcourt

Kool

2012

Nucleic Acids Research

111

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MicroRNAs (miRNAs) are a class of RNAs that play important regulatory roles in the cell. The detection of microRNA has attracted significant interest recently, as abnormal miRNA expression has been linked to cancer and other diseases. Here, we present a straightforward method for isothermal amplified detection of miRNA that involves two separate nucleic acid-templated chemistry steps. The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5′-iodide and a 3′-phosphorothioate. The sequence is amplified through rolling circle amplification with ϕ29 DNA polymerase and then detected via a second amplification using fluorogenic templated probes. Tests showed that the cyclization proceeds in ∼50% yield over 24 h and is compatible with the conditions required for rolling circle polymerization, unlike enzymatic ligations which required non-compatible buffer conditions. The polymerization yielded 188-fold amplification, and separate experiments showed ∼15-fold signal amplification from the templated fluorogenic probes. When all components are combined, results show miRNA detection down to 200 pM in solution, and correlation of the detected signal with the initial concentration of miRNA. The doubly templated double-amplification method demonstrates a new approach to detection of rolling circle products and significant advantages in ease of operation for miRNA detection.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Amplified microRNA detection by templated chemistry

Harcourt

Kool

2012

Nucleic Acids Research

111

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“…For a specific analysis of circularization, the concatemer formed would have to be significantly larger than any copies of the single‐stranded RNA formed. To achieve the most effective differentiation possible, we opted for the branched rolling‐circle amplification (BRCA) format, in which the initially formed cDNA is again amplified by a specific set of primers . We chose CRKL as an exemplary and—due to its length of 466 nucleotides—also challenging template.…”

Section: Figurementioning

confidence: 99%

A Fast and Easy Method for Specific Detection of Circular RNA by Rolling‐Circle Amplification

Boss

2019

ChemBioChem

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Circular RNAs (circRNAs) represent a new class of usually noncoding transcripts with largely unknown functions. Their research is hampered not least by the inapplicability of traditional analytical methods. Herein we describe a rapid and easy assay for the detection of natural circRNA, based on rolling‐circle amplification (RCA). This technique does not require the use of fluorescently labeled RNA or DNA and can specifically detect circular RNA in the presence of a 1000‐fold excess of the same linear RNA. Only standard devices such as (quantitative) PCR cyclers and gel electrophoresis are used.

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“…Nevertheless, its sensitivity and specificity for detecting miRNA are not satisfactory. Methodological improvement on the primers/probes design and label, chemical modification of nucleotides and signal amplification has been achieved with several recent developed nucleic acid detection strategies, such as pyreneexcimer probe [13] , rolling circle amplification [14,15] , doublytemplate-amplification [16] , enzyme conjugates [17] , lateral-flowbiosensors [18] , MB-mediated cycled polymerization [19] , and our proposed Ltail-CSDA methods [20,21] . These methods performed DNA detection sensitively, even with the PCR-like sensitivity [18,21] .…”

Section: Introductionmentioning

confidence: 99%

Ultrasensitive label-free detection of miRNA with asymmetric hairpin probe, exonuclease I and SYBR Green I

Liu

Long

et al. 2015

Chem. Res. Chin. Univ.

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Detection of miRNAs presents a particular challenge because of their limited size, high sequence homology and greatly various expression level. In this work, an ultrasensitive, label-free and isothermal miRNA detection was developed based on asymmertric hairpin probe, exonuclease I(Exo I) and SYBR Green I. The method employed asymmetric hairpin probe to perform cycled polymerization and Exo I to reduce background signal. In the presence of the target miRNA, the target triggers probe-mediated cycled polymerization reactions to generate lots of dsDNA products. The dsDNA product effectively prevents itself from being degraded by Exo I and permitted the insertion of more fluorescence dye into it to enlarge the fluorescence signal. In the absence of the target miRNA, there was no probe-mediated polymerization reaction, and the probe was digested by Exo I added, which minimized the intercalation of fluorescence dye to reduce the background signal. The combination of the probe-mediated cycled polymerization with the Exo I-assisted background reduction allows us to achieve a detection limit of 5×10 -18 mol/L. Owing to its ultrasensitivity, excellent specificity, convenience and low-cost, this method might hold out great promise in miRNA detection.

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Rolling‐Circle Amplification: Unshared Advantages in miRNA Detection

Cited by 32 publications

References 20 publications

Amplified microRNA detection by templated chemistry

Amplified microRNA detection by templated chemistry

A Fast and Easy Method for Specific Detection of Circular RNA by Rolling‐Circle Amplification

Ultrasensitive label-free detection of miRNA with asymmetric hairpin probe, exonuclease I and SYBR Green I

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