scholarly journals Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell.

1989 ◽  
Vol 37 (5) ◽  
pp. 697-701 ◽  
Author(s):  
F J Tang ◽  
P O Ts'o ◽  
S A Lesko
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
In Situ Hybridization ◽  
Single Cells ◽  
Specific Gene ◽  
Whole Cells ◽  
Beta Interferon ◽  
Ht1080 Cells ◽  
In Situ Detection

We report a quantitative method that combines in situ mRNA hybridization with microfluorometric analysis of DNA content to detect gene expression in single cells of a heteroploid cell population. The model was a human fibrosarcoma HT1080 cell line which consisted of diploid and tetraploid cells that were induced with polyI:polyC for production of beta-interferon. The level of beta-interferon mRNA detected by in situ hybridization was found to be two to three times higher in tetraploid compared to diploid HT1080 cells, and correlated with beta-interferon activity in that a subclone of tetraploid HT1080 cells secreted two- to fivefold more beta-interferon than a subclone of diploid HT1080 cells. Interestingly, beta-interferon-related transcripts were detected during S-phase in uninduced tetraploid HT1080 cells. In addition, beta-interferon induced by polyI:polyC was expressed in all phases of the cell cycle as demonstrated with a human diploid fibroblast, HF926. The unique features offered by the combination of microfluorometry and in situ hybridization provide a valuable tool to investigate specific gene expression related to ploidy or cell-cycle stage in the same individual cell of an unsynchronized population. Since the method allows direct observation of morphology, one can be assured that all quantitative measurements were made on whole cells with intact nuclei.

scholarly journals Gene expression analysis by non-radioactive RNA-RNA in situ hybridization techniques

2004 ◽  
Vol 23 (2) ◽  
pp. 127-133 ◽  
Author(s):  
Danijela Drakulic ◽  
Milena Stevanovic ◽  
Gordana Nikcevic
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Cultured Cells ◽  
Specific Gene ◽  
Rna In Situ Hybridization ◽  
Sox Gene ◽  
Labeled Rna ◽  
Set Up

RNA-RNA in situ hybridization is a reliable method for studying tissue and cell specific gene expression, which enables visualization of labeled antisense RNA probe hybridized to specific mRNA. In this study we employed non-radioactive RNA-RNA in situ hybridization using biotin- or digoxigenin-labeled RNA probes in order to detect SOX gene expression in carcinoma cell lines. By this approach we confirmed results obtained by Northern blot analysis, where the presence of SOX2 mRNA in NT2/D1 and SOX14 mRNA in HepG2 cells has been established. Our aim was to set up RNA-RNA in situ hybridization method in in vitro cultured cells in order to perform further analyses of SOX gene expression on various normal and cancer tissues.

Gene expression profiling of microdissected Hodgkin Reed-Sternberg cells correlates with treatment outcome in classical Hodgkin lymphoma

Blood ◽  
2012 ◽  
Vol 120 (17) ◽  
pp. 3530-3540 ◽  
Author(s):  
Christian Steidl ◽  
Arjan Diepstra ◽  
Tang Lee ◽  
Fong Chun Chan ◽  
Pedro Farinha ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Treatment Failure ◽  
Gene Expression Profiling ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Expression Profiling ◽  
Specific Gene ◽  
Classical Hodgkin Lymphoma

Abstract In classical Hodgkin lymphoma (CHL), 20%-30% of patients experience relapse or progressive disease after initial treatment. The pathogenesis and biology of treatment failure are still poorly understood, in part because the molecular phenotype of the rare malignant Hodgkin Reed-Sternberg (HRS) cells is difficult to study. Here we examined microdissected HRS cells from 29 CHL patients and 5 CHL-derived cell lines by gene expression profiling. We found significant overlap of HL-specific gene expression in primary HRS cells and HL cell lines, but also differences, including surface receptor signaling pathways. Using integrative analysis tools, we identified target genes with expression levels that significantly correlated with genomic copy-number changes in primary HRS cells. Furthermore, we found a macrophage-like signature in HRS cells that significantly correlated with treatment failure. CSF1R is a representative of this signature, and its expression was significantly associated with progression-free and overall survival in an independent set of 132 patients assessed by mRNA in situ hybridization. A combined score of CSF1R in situ hybridization and CD68 immunohistochemistry was an independent predictor for progression-free survival in multivariate analysis. In summary, our data reveal novel insights into the pathobiology of treatment failure and suggest CSF1R as a drug target of at-risk CHL.

scholarly journals Analysis of Gene Expression Patterns of Epigenetic Enzymes Dnmt3a, Tet1 and Ogt in Murine Chondrogenic Models

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2678
Author(s):  
Judit Vágó ◽  
Katalin Kiss ◽  
Edina Karanyicz ◽  
Roland Takács ◽  
Csaba Matta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
In Situ Hybridization ◽  
Marker Genes ◽  
Specific Gene ◽  
Cartilage Formation ◽  
Methylation Inhibitor ◽  
Dna Methylation Inhibitor

We investigated the gene expression pattern of selected enzymes involved in DNA methylation and the effects of the DNA methylation inhibitor 5-azacytidine during in vitro and in vivo cartilage formation. Based on the data of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3), and Ogt (O-linked N-acetylglucosamine transferase) were further examined with RT-qPCR in murine cell line-based and primary chondrifying micromass cultures. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton using specific RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine reduced cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory effect when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of key chondrogenic marker genes was altered by the treatment. Our results indicate that the DNA demethylation inducing Tet1 plays a significant role during chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

scholarly journals Single cells can sense their position in a morphogen gradient

Development ◽  
1999 ◽  
Vol 126 (23) ◽  
pp. 5309-5317 ◽  
Author(s):  
J.B. Gurdon ◽  
H. Standley ◽  
S. Dyson ◽  
K. Butler ◽  
T. Langon ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Single Cells ◽  
Morphogen Gradient ◽  
Rnase Protection ◽  
Lateral Contact ◽  
Related Response ◽  
Sense And Respond ◽  
Alternative Idea

Xenopus blastula cells show a morphogen-like response to activin by expressing different genes according to the concentration of activin to which they are exposed. To understand how cells recognize their position in a concentration gradient, it is essential to know whether each cell responds individually to activin concentration. An alternative idea, proposed by previous work, is that cells need to interact with their neighbours to generate a concentration-related response. To distinguish between these ideas, we have cultured blastula cells under conditions which provide different degrees of contact with other cells, allowing nil to maximum communication with their neighbours. The cultures include cells attached to fibronectin and cells resting unattached on an agarose base. The cultures also include cells that have no contact with any cell except their clonal progeny, cells that have lateral contact to neighbouring cells, and cells that are completely enveloped by other cells in a reaggregate. We have used RNase protection and in situ hybridization to assay the expression of the activin-responsive Xenopus genes Xbra, Xgsc, Xeomes, Xapod, Xchordin, Mix1, Xlim1 and Cerberus. We find no difference in gene expression between cells attached to fibronectin and those unattached on agarose. Most importantly, we find that cells respond to activin in a concentration-related way irrespective of their degree of contact with other cells. Therefore interaction among cells is not required for the interpretation of morphogen concentration, at least in the case of the early genes studied here. We conclude that isolated blastula cells can sense and respond individually to activin by expressing genes in a concentration-dependent way.

scholarly journals A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

2017 ◽  
Author(s):  
Chiara Sinigaglia ◽  
Daniel Thiel ◽  
Andreas Hejnol ◽  
Evelyn Houliston ◽  
Lucas Leclère
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Developmental Regulation ◽  
Expression Patterns ◽  
Urea Solution ◽  
Similar Reduction ◽  
In Situ Detection ◽  
Precise Expression ◽  
Significant Health

AbstractIn situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has proven to be essential to our understanding of biological processes, including developmental regulation. In situ protocols are today routine in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acids strand. This step, in general, is carried out at high temperatures and in a denaturing solution, the hybridization buffer, commonly containing 50% (v/v) formamide. An important drawback is that hot formamide poses a significant health risk and so must be handled with great care.We were prompted to test alternative hybridization solutions for in situ detection of gene expression in the medusa of the hydrozoan Clytia hemisphaerica, where traditional protocols caused extensive deterioration of the morphology and texture during hybridization, hindering observation and interpretation of results. Inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8 M urea solution in the hybridization buffer. The new protocol yielded better morphologies and consistency of tissues, and also notably improved the resolution of the signal, allowing more precise localization of gene expression, as well as reduced staining at non-specific sites. Given the improved results using a less toxic hybridization solution, we tested the urea protocol on a number of other metazoans: two brachiopod species (Novocrania anomala and Terebratalia transversa) and the worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea in in situ hybridization offers safer alternative protocols, potentially useful in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

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