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

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.

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

Clinical Cancer Research ◽

10.1158/1078-0432.ccr-08-3257 ◽

2009 ◽

Vol 15 (12) ◽

pp. 4009-4016 ◽

Cited By ~ 121

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

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Diagnosis of Polyomavirus-Induced Hepatic Necrosis in Psittacine Birds Using DNA Probes

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879400600305 ◽

1994 ◽

Vol 6 (3) ◽

pp. 308-314 ◽

Cited By ~ 7

Author(s):

AnaPatricia Garcia ◽

Kenneth S. Latimer ◽

Frank D. Niagro ◽

Branson W. Ritchie ◽

Raymond P. Campagnoli

Keyword(s):

In Situ Hybridization ◽

Nucleic Acid ◽

Dna Amplification ◽

Hepatic Necrosis ◽

Dna Probes ◽

Intranuclear Inclusions ◽

Hepatocellular Necrosis ◽

Hematoxylin And Eosin ◽

Avian Polyomavirus

Liver sections from 32 psittacine birds with multifocal to coalescing hepatocellular necrosis were examined to determine the cause of disease. Avian polyomavirus (APV) infection (19 of 32 birds), bacterial hepatitis (5 of 32 birds), and chlamydiosis (3 of 32 birds) were major causes of hepatic disease. The presence of APV inclusions or nucleic acid was demonstrated using hematoxylin and eosin (HE) staining, DNA in situ hybridization, and DNA amplification with Southern blotting. Amphophilic intranuclear inclusions, suggestive of APV infection, were observed in HE-stained liver sections from 5 of 32 birds. Hepatocellular karyomegaly was present in liver tissues from 10 birds (5 birds with typical APV inclusions and 5 birds without discernable inclusions). DNA in situ hybridization recognized intranuclear APV nucleic acid in liver sections of 18 of 32 birds. DNA amplification with Southern or dot blots also identified APV nucleic acid in processed, paraffin-embedded livers of 18 of 32 birds. This study demonstrates that acute APV infection is a frequent cause of multifocal to coalescing hepatocellular necrosis in psittacine birds. Furthermore, APV infection is best diagnosed using DNA probes, especially when typical intranuclear inclusions are not observed microscopically.

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