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Amplified microRNA detection by templated chemistry.

Harcourt EM, Kool ET - Nucleic Acids Res. (2012)

Bottom Line: The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5'-iodide and a 3'-phosphorothioate.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.

ABSTRACT
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 29 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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Schematic illustration of miRNA detection design. Autoligation of an activated circle precursor (grey) in the presence of an analyte template (blue) is followed by RCA; the rolling circle product is detected through fluorescence turn-on of sequence-specific Q-STAR probes.
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gkr1313-F1: Schematic illustration of miRNA detection design. Autoligation of an activated circle precursor (grey) in the presence of an analyte template (blue) is followed by RCA; the rolling circle product is detected through fluorescence turn-on of sequence-specific Q-STAR probes.

Mentions: With these considerations in mind, we have devised an isothermal double amplification technique for miRNA detection consisting of (i) RNA template-dependent autoligation of a circle precursor; (ii) RCA using ϕ29 DNA polymerase and primed by the RNA sequence and (iii) fluorescence detection of the rolling circle products using Q-STAR probes (Figure 1). Here, we describe the development of the methods and probes and their performance in the detection of let-7a RNA.Figure 1.


Amplified microRNA detection by templated chemistry.

Harcourt EM, Kool ET - Nucleic Acids Res. (2012)

Schematic illustration of miRNA detection design. Autoligation of an activated circle precursor (grey) in the presence of an analyte template (blue) is followed by RCA; the rolling circle product is detected through fluorescence turn-on of sequence-specific Q-STAR probes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351153&req=5

gkr1313-F1: Schematic illustration of miRNA detection design. Autoligation of an activated circle precursor (grey) in the presence of an analyte template (blue) is followed by RCA; the rolling circle product is detected through fluorescence turn-on of sequence-specific Q-STAR probes.
Mentions: With these considerations in mind, we have devised an isothermal double amplification technique for miRNA detection consisting of (i) RNA template-dependent autoligation of a circle precursor; (ii) RCA using ϕ29 DNA polymerase and primed by the RNA sequence and (iii) fluorescence detection of the rolling circle products using Q-STAR probes (Figure 1). Here, we describe the development of the methods and probes and their performance in the detection of let-7a RNA.Figure 1.

Bottom Line: The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5'-iodide and a 3'-phosphorothioate.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.

ABSTRACT
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 29 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.

Show MeSH
Related in: MedlinePlus