Cell-specific metallothionein gene expression in mouse decidua and placentae

Oligodeoxyribonucleotide excess solution hybridization, Northern blot and in situ hybridization were used to analyze metallothionein gene expression in mouse decidua and placentae during gestation. Metallothionein (MT) -I and -II mRNA levels were constitutively elevated, 11- and 13-fold, respectively, relative to the adult liver, in the deciduum (D8), and decreased coordinately about 6-fold during the period of development when the deciduum is replaced by the developing placenta (D10-16). Coincident with this decline, levels of MT mRNA increased dramatically in the visceral yolk sac endoderm. In situ hybridization established that MT-I mRNA was present at low levels in the uterine luminal epithelium (D4), but was elevated at the site of embryo implantation exclusively in the primary decidual zone by D5, and then in the secondary decidual zone (D6-8). Although low levels of MT mRNA were detected in total placental RNA, in situ hybridization revealed constitutively high levels in the outer placental spongiotrophoblasts. Analysis of pulse-labeled proteins from decidua and placentae established that these tissues are active in the synthesis of MT. The constitutively high levels of MT mRNA in decidua were only slightly elevated following injection of cadmium (Cd) and/or zinc (Zn), whereas in placentae they increased several-fold. MT mRNA levels were equally high in decidua and experimentally induced deciduomata (D8) which establishes that decidual MT gene expression is not dependent on the presence of the embryo or some embryo-derived factor. Although the functional role of MT during development is speculative, these results establish the concept that, from the time of implantation to late in gestation, the mouse embryo is surrounded by cells, interposed between the maternal and embryonic environments, which actively express the MT genes. This suggests that MT plays an important role in the establishment and maintenance of normal pregnancy.

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RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

Methods and Protocols ◽

10.3390/mps4010020 ◽

2021 ◽

Vol 4 (1) ◽

pp. 20

Author(s):

Mujeeb Shittu ◽

Tessa Steenwinkel ◽

William Dion ◽

Nathan Ostlund ◽

Komal Raja ◽

...

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Expression Patterns ◽

Drosophila Species ◽

Gene Expression Patterns ◽

Probe Design ◽

Tissue Preparation ◽

Design And Synthesis ◽

Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

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Methods to Enhance Signal Using Isotopic In Situ Hybridization

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540205000805 ◽

2002 ◽

Vol 50 (8) ◽

pp. 1031-1037 ◽

Cited By ~ 13

Author(s):

Betty Ky ◽

Paul J. Shughrue

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Cell Types ◽

Oxytocin Receptor ◽

Specific Cell ◽

Rat Hypothalamus ◽

Low Levels ◽

Tuberoinfundibular Peptide ◽

Cryostat Sections

Isotopic in situ hybridization (ISH) has been established as a uniquely powerful tool for the study of gene expression in specific cell types. This technique allows the visualization and quantification of gene expression and gene expression changes in cells. In our study of biological and molecular phenomena, we have increasingly encountered the need to detect small changes in gene expression as well as genes of low abundance, such as the oxytocin receptor (OTR) and the tuberoinfundibular peptide of 39 residues (Tip39). To increase the sensitivity of isotopic ISH for detection of rare mRNAs, we performed ISH on cryostat sections of rat hypothalamus and thalamus with 35S-labeled riboprobes and amplified the signal by hybridizing over 2 nights as well as labeling the probe with both [35S]-UTP and [35S]-ATP. These two methods of enhancement independently and in combination demonstrated a dramatic increase in signal, allowing the visualization of low levels of gene expression previously undetectable by conventional methods.

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

Jugoslovenska medicinska biohemija ◽

10.2298/jmh0402127d ◽

2004 ◽

Vol 23 (2) ◽

pp. 127-133 ◽

Cited By ~ 1

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.

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Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

Nucleic Acids Research ◽

10.1093/nar/gkz1151 ◽

2019 ◽

Vol 48 (3) ◽

pp. e17-e17 ◽

Cited By ~ 2

Author(s):

Lena Voith von Voithenberg ◽

Anna Fomitcheva Khartchenko ◽

Deborah Huber ◽

Peter Schraml ◽

Govind V Kaigala

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Spatial Heterogeneity ◽

Tumor Heterogeneity ◽

Expression Patterns ◽

Spatial Multiplexing ◽

Local Analysis ◽

Tissue Sections ◽

Rna In Situ Hybridization

Abstract Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases.

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Application of Nonisotopic RNA In Situ Hybridization to Detect Endogenous Gene Expression in Pathologic Specimens

Diagnostic Molecular Pathology ◽

10.1097/00019606-199303000-00037 ◽

1993 ◽

Vol 2 (1) ◽

pp. 277-282

Author(s):

Mabel Pang ◽

Linda G. Baum

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Endogenous Gene ◽

Rna In Situ Hybridization

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Presence and distribution of endothelin-1 gene expression in human kidney

AJP Renal Physiology ◽

10.1152/ajprenal.1994.267.4.f679 ◽

1994 ◽

Vol 267 (4) ◽

pp. F679-F687 ◽

Cited By ~ 9

Author(s):

C. Pupilli ◽

M. Brunori ◽

N. Misciglia ◽

C. Selli ◽

L. Ianni ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

In Situ Hybridization ◽

Renal Cortex ◽

Cdna Probe ◽

Renal Medulla ◽

Human Kidney ◽

Renal Tumors ◽

Mrna Levels

To investigate the presence and the distribution of preproendothelin-1 (prepro-ET-1) mRNA in human kidney, eight human kidneys obtained at surgery from patients affected by localized renal tumors were studied. Northern blot analysis using a human prepro-ET-1 cDNA probe labeled with 32P showed the presence of a single band of approximately 2.3 kb that was present both in the renal cortex and medulla of all the kidneys studied. Densitometric analysis of hybridization signals demonstrated that prepro-ET-1 mRNA levels in the renal medulla were 2.2-fold higher than those in the renal cortex. The distribution of prepro-ET-1 mRNA in human kidney was investigated by in situ hybridization using a human prepro-ET-1 RNA probe labeled with 35S. The greatest density of prepro-ET-1 mRNA was observed in the renal medulla, where hybridization signal was demonstrated in vasa recta bundles and capillaries and in collecting ducts. By combining in situ hybridization with immunohistochemical detection of von Willebrand factor, we demonstrated that 93 +/- 2.5% of nontubular medullary cells containing prepro-ET-1 mRNA were endothelial cells. In the cortex, prepro-ET-1 mRNA was localized in the endothelial layer of arcuate and interlobular arteries and veins and in the endothelial cells of afferent arterioles. The results of the present study demonstrate that ET-1 gene expression is present in vascular and tubular structures of the human kidney. It is possible that ET-1 synthesized locally in the human kidney represents a local system affecting renal hemodynamics and functions through paracrine and/or autocrine actions on different renal structures.

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Analysis of AC3-33 gene expression in multiple organ cancer and adjacent normal tissue by RNA in situ hybridization

Oncology Letters ◽

10.3892/ol.2015.3112 ◽

2015 ◽

Vol 9 (6) ◽

pp. 2795-2798

Author(s):

FEN HU ◽

SHAOQING YANG ◽

SHAOBO LV ◽

YAN PENG ◽

LIJUN MENG ◽

...

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Normal Tissue ◽

Multiple Organ ◽

Adjacent Normal Tissue ◽

Rna In Situ Hybridization

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Localization of protamine 1 mRNA in different stages of the cycle of the rat seminiferous epithelium.

The Journal of Cell Biology ◽

10.1083/jcb.107.2.407 ◽

1988 ◽

Vol 107 (2) ◽

pp. 407-412 ◽

Cited By ~ 33

Author(s):

P Mali ◽

M Sandberg ◽

E Vuorio ◽

P C Yelick ◽

N B Hecht ◽

...

Keyword(s):

In Situ Hybridization ◽

Northern Blot ◽

Northern Blot Analysis ◽

Cdna Probe ◽

Seminiferous Epithelium ◽

Seminiferous Tubules ◽

Mrna Levels ◽

Low Levels ◽

Protamine 1

A mouse protamine 1 cDNA probe was used to study P1 protamine gene expression during the cycle of the seminiferous epithelium in the rat. In situ hybridization experiments showed that transcription of the P1 protamine mRNA starts in the middle of step 7 of spermiogenesis during substage VIIc. The mRNA levels stay high in steps 7-14 spermatids but decrease during steps 15-16 and are virtually undetectable in steps 17-19 spermatids. Northern blot analyses of RNAs isolated from microdissected pools of seminiferous tubules show high P1 protamine mRNA concentrations during stages VIIc-XIV-III of the cycle and lower levels during stages IV-VIIb. Owing to a post-transcriptional shortening of the poly(A) tail by 130 bases, a decrease in the size of protamine 1 mRNA from approximately 580 to 450 nucleotides was observed in stages XIII-XIV suggesting an initiation of protamine 1 synthesis in step 13-14 spermatids. In stages II-VI (steps 16-18 spermatids), only the smaller size protamine 1 mRNA was detectable. The expression of protamine 1 mRNAs has been localized in the very last phase of the haploid gene activity. Although the in situ hybridization suggests a disappearance of protamine 1 mRNA after step 16 of spermiogenesis, Northern blot analysis shows that low levels of mRNA are present during the period of final condensation of the chromatin, reflecting the association of protamine with DNA.

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