Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences

Matthiesen, Steen H.; Hansen, Claus Thorp

doi:10.1371/journal.pone.0040675

Cited by 70 publications

(67 citation statements)

References 31 publications

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“…To analyze the relationship between different types of probes and specificity, we performed melting studies for all probes with DNA and RNA targets. The use of different tar-gets is important because even though the FISH methodology is more frequently described for RNA targets, there are also several studies using modified probes which use DNA as a target, e.g., (Celeda et al 1994;Matthiesen and Hansen 2012). In accordance with the literature, a higher Tm was observed at higher salt-concentrated solutions (Owczarzy et al 2004).…”

Section: Discussionmentioning

confidence: 53%

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Fontenete

Barros

Madureira

et al. 2015

Appl Microbiol Biotechnol

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In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is per-formed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids 2 (LNA)/2′-O-methyl RNA (2′OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2′OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

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Section: Discussionmentioning

confidence: 53%

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Fontenete

Barros

Madureira

et al. 2015

Appl Microbiol Biotechnol

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“…Lysates were homogenized through a narrow gauge needle (0.4 mm diameter). After prewarming the lysate to 60°C, 15% v/v ethylenecarbonate (Sigma-Aldrich) was added as a hybridization enhancer (Matthiesen and Hansen 2012). Three-hundred microliters LNA/ DNA mixmer oligonucleotide coupled to magnetic beads, prepared as described above, were added to the lysate and incubated for 2 h at 41°C, inverting the tubes every 15 min, four times.…”

Section: Specific Rnp Capture In Hela Cellsmentioning

confidence: 99%

Specific RNP capture with antisense LNA/DNA mixmers

Rogell

Fischer

Rettel

et al. 2017

RNA

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RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

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“…We also note the value of bright-field ISH techniques (ie, CISH, SISH, BDISH, and DDISH) in assessment of genetic heterogeneity, as these assays permit evaluation of the entire slide under light microscopy. 72 Improved FISH techniques 87 may also reduce the risk of overlooking focal amplified cells due to signal quality and increased DAPI counterstain. In addition, staining of slides can be carried out within 4 hours (J Rü schoff, in preparation).…”

Section: Authors' Consensus Recommendationsmentioning

confidence: 99%

HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity

Hanna

Rüschoff²,

Bilous³

et al. 2014

Modern Pathology

252

201

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Trastuzumab-containing therapy is a standard of care for patients with HER2 þ breast cancer. HER2 status is routinely assigned using in situ hybridization to assess HER2 gene amplification, but interpretation of in situ hybridization results may be challenging in tumors with chromosome 17 polysomy or intratumoral genetic heterogeneity. Apparent chromosome 17 polysomy, defined by increased chromosome enumeration probe 17 (CEP17) signal number, is a common genetic aberration in breast cancer and represents an alternative mechanism for increasing HER2 copy number. Some studies have linked elevated CEP17 count ('polysomy') with adverse clinicopathologic features and HER2 overexpression, although there are numerous discrepancies in the literature. There is evidence that elevated CEP17 ('polysomy') count might account for trastuzumab response in tumors with normal HER2:CEP17 ratios. Nonetheless, recent studies establish that apparent 'polysomy' (CEP17 increase) is usually related to focal pericentromeric gains rather than true polysomy. Assigning HER2 status may also be complex where multiple cell subclones with distinct HER2 amplification characteristics coexist within the same tumor. Such genetic heterogeneity affects up to 40% of breast cancers when assessed according to a College of American Pathologists guideline, although other definitions have been proposed. Recent data have associated heterogeneity with unfavorable clinicopathologic variables and poor prognosis. Genetically heterogeneous tumors harboring HER2-amplified subclones have the potential to benefit from trastuzumab, but this has yet to be evaluated in clinical studies. In this review, we discuss the implications of apparent polysomy 17 and genetic heterogeneity for assigning HER2 status in clinical practice. Among our recommendations, we support the use of mean HER2 copy number rather than HER2:CEP17 ratio to define HER2 positivity in cases where coamplification of the centromere might mask HER2 amplification. We also highlight a need to harmonize in situ hybridization scoring methodology to support accurate HER2 status determination, particularly where there is evidence of heterogeneity.

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Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences

Cited by 70 publications

References 31 publications

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Specific RNP capture with antisense LNA/DNA mixmers

HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity

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