Achieving Single-Nucleotide Specificity in Direct Quantitative Analysis of Multiple MicroRNAs (DQAMmiR)

2016 ◽  
Vol 88 (4) ◽  
pp. 2472-2477 ◽  
Author(s):  
David W. Wegman ◽  
Farhad Ghasemi ◽  
Alexander S. Stasheuski ◽  
Anna Khorshidi ◽  
Burton B. Yang ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Single Nucleotide ◽  
Nucleotide Specificity

scholarly journals Cellular microRNA detection with miRacles: microRNA- activated conditional looping of engineered switches

2019 ◽  
Vol 5 (3) ◽  
pp. eaau9443 ◽  
Author(s):  
Arun Richard Chandrasekaran ◽  
Molly MacIsaac ◽  
Paromita Dey ◽  
Oksana Levchenko ◽  
Lifeng Zhou ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Northern Blotting ◽  
Single Step ◽  
Single Nucleotide ◽  
Disease Biomarkers ◽  
Microrna Detection ◽  
Detection Assay ◽  
Polymerase Chain ◽  
Regulatory Rnas ◽  
Nucleotide Specificity

MicroRNAs are short noncoding regulatory RNAs that are increasingly used as disease biomarkers. Detection of microRNAs can be arduous and expensive and often requires amplification, labeling, or radioactive probes. Here, we report a single-step, nonenzymatic microRNA detection assay using conformationally responsive DNA nanoswitches. Termed miRacles (microRNA-activated conditional looping of engineered switches), our assay has subattomole sensitivity and single-nucleotide specificity using an agarose gel electrophoresis readout. We detect cellular microRNAs from nanogram-scale RNA extracts of differentiating muscle cells and multiplex our detection for several microRNAs from one biological sample. We demonstrate 1-hour detection without expensive equipment or reagents, making this assay a compelling alternative to quantitative polymerase chain reaction and Northern blotting.

scholarly journals Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR34/L98H- and TR46/Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

2015 ◽  
Vol 60 (1) ◽  
pp. 387-392 ◽  
Author(s):  
Faezeh Mohammadi ◽  
Seyed Jamal Hashemi ◽  
Jan Zoll ◽  
Willem J. G. Melchers ◽  
Haleh Rafati ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Quantitative Analysis ◽  
Single Nucleotide Polymorphisms ◽  
Aspergillus Fumigatus ◽  
Tandem Repeat ◽  
Promoter Region ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type

ABSTRACTWe employed an endpoint genotyping method to update the prevalence rate of positivity for the TR34/L98H mutation (a 34-bp tandem repeat mutation in the promoter region of thecyp51Agene in combination with a substitution at codon L98) and the TR46/Y121F/T289A mutation (a 46-bp tandem repeat mutation in the promoter region of thecyp51Agene in combination with substitutions at codons Y121 and T289) among clinicalAspergillus fumigatusisolates obtained from different regions of Iran over a recent 5-year period (2010 to 2014). The antifungal activities of itraconazole, voriconazole, and posaconazole against 172 clinicalA. fumigatusisolates were investigated using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. For the isolates with an azole resistance phenotype, thecyp51Agene and its promoter were amplified and sequenced. In addition, using a LightCycler 480 real-time PCR system, a novel endpoint genotyping analysis method targeting single-nucleotide polymorphisms was evaluated to detect the L98H and Y121F mutations in thecyp51Agene of all isolates. Of the 172A. fumigatusisolates tested, the MIC values of itraconazole (≥16 mg/liter) and voriconazole (>4 mg/liter) were high for 6 (3.5%). Quantitative analysis of single-nucleotide polymorphisms showed the TR34/L98H mutation in thecyp51Agenes of six isolates. No isolates harboring the TR46/Y121F/T289A mutation were detected. DNA sequencing of thecyp51Agene confirmed the results of the novel endpoint genotyping method. By microsatellite typing, all of the azole-resistant isolates had genotypes different from those previously recovered from Iran and from the Dutch TR34/L98H controls. In conclusion, there was not a significant increase in the prevalence of azole-resistantA. fumigatusisolates harboring the TR34/L98H resistance mechanism among isolates recovered over a recent 5-year period (2010 to 2014) in Iran. A quantitative assay detecting a single-nucleotide polymorphism in thecyp51Agene ofA. fumigatusis a reliable tool for the rapid screening and monitoring of TR34/L98H- and TR46/Y121F/T289A-positive isolates and can easily be incorporated into clinical mycology algorithms.

Chemo-biological mRNA imaging with single nucleotide specificity

2019 ◽  
Vol 55 (98) ◽  
pp. 14817-14820 ◽  
Author(s):  
Andrea Knoll ◽  
Svenja Kankowski ◽  
Sophie Schöllkopf ◽  
Jochen C. Meier ◽  
Oliver Seitz
Keyword(s):  
Cellular Imaging ◽  
Rna Detection ◽  
Single Nucleotide ◽  
Imaging Methods ◽  
Combined Use ◽  
Rna Imaging ◽  
Nucleotide Specificity

The combined use of “biological” RNA imaging methods based on MS2 technology and “chemical” RNA detection by FIT probes allows unambiguous cellular imaging of a C → U edit in mRNA encoding for GlyR α2.

scholarly journals Droplet-digital Cas13a assay enables direct single-molecule microRNA quantification

2019 ◽  
Author(s):  
Tian Tian ◽  
Bowen Shu ◽  
Lei Liu ◽  
Xiaoming Zhou
Keyword(s):  
Cell Lines ◽  
Single Molecule ◽  
Absolute Quantification ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Broad Applicability ◽  
Precise Quantification ◽  
Nucleotide Specificity ◽  
Direct Quantification

The direct quantification of microRNA at the single-molecule level is still challenging. Herein, we developed a droplet-digital Cas13a assay (ddCA) as a general approach for the amplification-free and absolute quantification of single unlabeled miRNA molecules with single-nucleotide specificity. We demonstrate its simplicity, precise quantification capability, excellent specificity and broad applicability by analyzing microRNAs from synthetic and cell lines-derived materials.

scholarly journals Single-base mapping of m6A by an antibody-independent method

2019 ◽  
Author(s):  
Zhang Zhang ◽  
Li-Qian Chen ◽  
Yu-Li Zhao ◽  
Cai-Guang Yang ◽  
Ian A Roundtree ◽  
...  
Keyword(s):  
High Throughput ◽  
High Reliability ◽  
Methylation Status ◽  
Independent Method ◽  
Location Information ◽  
Single Nucleotide ◽  
Single Base ◽  
Identification Method ◽  
Nucleotide Specificity ◽  
Base Mapping

AbstractN6-methyladenosine (m6A) is one of the most abundant mRNA modifications in eukaryotes, involved in various pivotal processes of RNA metabolism. The most popular high-throughput m6A identification method depends on the anti-m6A antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery and functions of m6A. Here we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease (m6A-sensitive RNA-Endoribonuclease-Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic, single-base m6A maps generated by m6A-REF-seq quantitatively displayed an explicit distribution pattern with enrichment on stop codons. Independent methods were used to validate the methylation status and abundance of individual m6A sites, confirming the high reliability and accuracy of m6A-REF-seq. We applied this method on five tissues from human, mouse and rat, showing that m6A sites were conserved with single nucleotide specificity and tend to cluster among species.

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