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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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Related in: MedlinePlus

Quantification of rolling circle copies from initial miRNA concentration. Autoligation and RCA were carried out simultaneously with 0 nM, 0.1 nM, 0.2 nM, 0.5 nM, 1 nM or 2 nM of RNA target for 24 h. After enzyme denaturation, 400 nM Q-STAR and 1.2 μM TPP were added. (A) Fluorescence traces with long QSTAR probes. (B) For the samples that gave a signal above background, the fluorescence signal was determined after 200 min and compared to the standard curve to calculate the concentration of circle copies. Error bars represent the standard deviation from six trials.
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gkr1313-F5: Quantification of rolling circle copies from initial miRNA concentration. Autoligation and RCA were carried out simultaneously with 0 nM, 0.1 nM, 0.2 nM, 0.5 nM, 1 nM or 2 nM of RNA target for 24 h. After enzyme denaturation, 400 nM Q-STAR and 1.2 μM TPP were added. (A) Fluorescence traces with long QSTAR probes. (B) For the samples that gave a signal above background, the fluorescence signal was determined after 200 min and compared to the standard curve to calculate the concentration of circle copies. Error bars represent the standard deviation from six trials.

Mentions: Once a standard curve had been created, it could be used to quantify products from our miRNA-templated ligation and RCA. Known concentrations of miRNA were incubated under ligation-RCA conditions for 24 h, at which point the probes were added. The fluorescent signal was recorded 200 min after addition of the probes and compared to that of the templated standards. Data showed that miRNA concentrations greater than 2 nM generated more than 400 nM of probe binding sites through ligation-RCA, and thus could not be quantified with the 400 nM of Q-STAR probe used. Concentrations of miRNA lower than 0.2 nM could not be distinguished from background. Thus, under this set of conditions the use of the long (non-turnover) Q-STAR probes gave a 10-fold sensitivity range for quantitative detection of miRNA (Figure 5). However, adjusting the probe concentration would change the range of concentrations of miRNA that can be quantitated.Figure 5.


Amplified microRNA detection by templated chemistry.

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

Quantification of rolling circle copies from initial miRNA concentration. Autoligation and RCA were carried out simultaneously with 0 nM, 0.1 nM, 0.2 nM, 0.5 nM, 1 nM or 2 nM of RNA target for 24 h. After enzyme denaturation, 400 nM Q-STAR and 1.2 μM TPP were added. (A) Fluorescence traces with long QSTAR probes. (B) For the samples that gave a signal above background, the fluorescence signal was determined after 200 min and compared to the standard curve to calculate the concentration of circle copies. Error bars represent the standard deviation from six trials.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1313-F5: Quantification of rolling circle copies from initial miRNA concentration. Autoligation and RCA were carried out simultaneously with 0 nM, 0.1 nM, 0.2 nM, 0.5 nM, 1 nM or 2 nM of RNA target for 24 h. After enzyme denaturation, 400 nM Q-STAR and 1.2 μM TPP were added. (A) Fluorescence traces with long QSTAR probes. (B) For the samples that gave a signal above background, the fluorescence signal was determined after 200 min and compared to the standard curve to calculate the concentration of circle copies. Error bars represent the standard deviation from six trials.
Mentions: Once a standard curve had been created, it could be used to quantify products from our miRNA-templated ligation and RCA. Known concentrations of miRNA were incubated under ligation-RCA conditions for 24 h, at which point the probes were added. The fluorescent signal was recorded 200 min after addition of the probes and compared to that of the templated standards. Data showed that miRNA concentrations greater than 2 nM generated more than 400 nM of probe binding sites through ligation-RCA, and thus could not be quantified with the 400 nM of Q-STAR probe used. Concentrations of miRNA lower than 0.2 nM could not be distinguished from background. Thus, under this set of conditions the use of the long (non-turnover) Q-STAR probes gave a 10-fold sensitivity range for quantitative detection of miRNA (Figure 5). However, adjusting the probe concentration would change the range of concentrations of miRNA that can be quantitated.Figure 5.

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