scholarly journals Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

10.3791/3655 ◽  
2012 ◽  
Author(s):  
Kelly L. Robertson ◽  
Gary J. Vora
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Small Rna ◽  
Locked Nucleic Acid ◽  
Rna Detection ◽  
Bacterial Small Rna

scholarly journals Locked Nucleic Acid and Flow Cytometry-FluorescenceIn SituHybridization for the Detection of Bacterial Small Noncoding RNAs

2011 ◽  
Vol 78 (1) ◽  
pp. 14-20 ◽  
Author(s):  
Kelly L. Robertson ◽  
Gary J. Vora
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Stationary Phase ◽  
Noncoding Rnas ◽  
Locked Nucleic Acid ◽  
Lag Phase ◽  
Bacterial Cells ◽  
Small Noncoding Rnas ◽  
Qrt Pcr

ABSTRACTWe describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescencein situhybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) inVibrio campbelliiATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter. Importantly, while LNA flow-FISH and qRT-PCR demonstrated similar overall expression trends, only LNA flow-FISH, which enabled the detection of ncRNAs in individual cells as opposed to the lysate-based ensemble measurements generated by qRT-PCR, was able to capture the cell-to-cell heterogeneity in ncRNA expression. As such, this study demonstrates a new method that simultaneously enables thein situdetection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population.

scholarly journals Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

2009 ◽  
Vol 15 (12) ◽  
pp. 4009-4016 ◽  
Author(s):  
Nobutake Yamamichi ◽  
Ryoichi Shimomura ◽  
Ken-ichi Inada ◽  
Kouhei Sakurai ◽  
Takeshi Haraguchi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Hybridization Analysis ◽  
Locked Nucleic Acid ◽  
Cancer Development

scholarly journals Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes

2006 ◽  
Vol 72 (8) ◽  
pp. 5311-5317 ◽  
Author(s):  
Kengo Kubota ◽  
Akiyoshi Ohashi ◽  
Hiroyuki Imachi ◽  
Hideki Harada
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Fluorescence Intensity ◽  
Dna Probes ◽  
Locked Nucleic Acid ◽  
Factors Affecting ◽  
Hybridization Efficiency ◽  
Probe Hybridization ◽  
Major Factors

ABSTRACT Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.

Distinguishing L from M photopigment coding sequences by hybridization to novel locked nucleic acid (LNA) oligonucleotide probes

2008 ◽  
Vol 25 (3) ◽  
pp. 283-287
Author(s):  
CHRISTINA PETTAN-BREWER ◽  
LI FU ◽  
SAMIR S. DEEB
Keyword(s):  
Nucleic Acid ◽  
Locked Nucleic Acid ◽  
Macaca Nemestrina ◽  
Oligonucleotide Probes ◽  
Coding Sequences ◽  
Cone Mosaic ◽  
Target Nucleic Acid ◽  
High Degree

Many attempts have been made over the years to distinguish human and primate L (long-wavelength sensitive) from M (middle-wavelength sensitive) cone photoreceptors using either immunohistochemistry or in situ hybridization. These attempts have been unsuccessful due to the very high degree of identity between the sequences of the L and M proteins and encoding mRNAs. The recent development of chemically modified oligonucleotide probes, referred to as locked nucleic acid (LNA) probes, has shown that they hybridize with much greater affinity and specificity to the target nucleic acid. This has greatly increased the potential for differentiating L from M cones by in situ hybridization. We have designed LNA oligonucleotide probes that are complementary to either the L or M coding sequences located in exon 5 of the Macaca nemestrina L and M pigment genes. We have shown that the LNA-M and LNA-L probes hybridize specifically to their respective target nucleic acid sequences in vitro. This result strongly suggests that these probes would be instrumental in rapidly distinguishing L from M cone in the entire retina, and in defining the cone mosaic during development and in adults.

scholarly journals Small RNA Detection by in Situ Hybridization Methods

2015 ◽  
Vol 16 (12) ◽  
pp. 13259-13286 ◽  
Author(s):  
Martyna Urbanek ◽  
Anna Nawrocka ◽  
Wlodzimierz Krzyzosiak
Keyword(s):  
In Situ Hybridization ◽  
Small Rna ◽  
Rna Detection

scholarly journals Optimizing locked nucleic acid/2’-O-methyl-RNA fluorescence in situ hybridization (LNA/2’OMe-FISH) procedure for bacterial detection

PLoS ONE ◽  
2019 ◽  
Vol 14 (5) ◽  
pp. e0217689 ◽  
Author(s):  
Andreia S. Azevedo ◽  
Inês M. Sousa ◽  
Ricardo M. Fernandes ◽  
Nuno F. Azevedo ◽  
Carina Almeida
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Fluorescence In Situ Hybridization ◽  
Locked Nucleic Acid ◽  
Bacterial Detection

scholarly journals Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Biofouling ◽  
2016 ◽  
Vol 32 (2) ◽  
pp. 179-190 ◽  
Author(s):  
Diana Vilas Boas ◽  
Carina Almeida ◽  
Sanna Sillankorva ◽  
Ana Nicolau ◽  
Joana Azeredo ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Fluorescent In Situ Hybridization ◽  
Locked Nucleic Acid ◽  
Infected Cells
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