scholarly journals Towards Quantitative In Situ Hybridization

1997 ◽  
Vol 45 (3) ◽  
pp. 413-423 ◽  
Author(s):  
Ard Jonker ◽  
Piet A. J. de Boer ◽  
Maurice J. B. van den Hoff ◽  
Wouter H. Lamers ◽  
Antoon F. M. Moorman
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Photographic Emulsion ◽  
Mrna Levels ◽  
Intestinal Tissue ◽  
Tissue Sections ◽  
Silver Grains

In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four- to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissue. (J Histochem Cytochem 45:413–423, 1997)

scholarly journals Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization.

1989 ◽  
Vol 108 (5) ◽  
pp. 1823-1832 ◽  
Author(s):  
O Horovitz ◽  
D Knaack ◽  
T R Podleski ◽  
M M Salpeter
Keyword(s):  
Ascorbic Acid ◽  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Alpha Subunit ◽  
Mrna Levels ◽  
Surface Receptors ◽  
Subunit Mrna

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.

Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel

1997 ◽  
Vol 272 (4) ◽  
pp. C1335-C1344 ◽  
Author(s):  
C. Ding ◽  
E. D. Potter ◽  
W. Qiu ◽  
S. L. Coon ◽  
M. A. Levine ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Pineal Gland ◽  
Northern Blot ◽  
Blot Analysis ◽  
Cyclic Nucleotide ◽  
Northern Blot Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

We used Northern blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction, and in situ hybridization to investigate the hypothesis that the CNG1 isoform of the cyclic nucleotide-gated nonselective cation channel may be widely distributed in tissues of the rat. A cDNA encoding the CNG1 isoform was isolated from rat eye and human retina, and partial sequences were isolated from rat pineal gland and human kidney. Northern blot analysis revealed a 3.1-kilobase (kb) CNG1 transcript in rat eye, pineal gland, pituitary, adrenal gland, and spleen, and a larger transcript of 3.5 kb was found in testis. RPA confirmed the identity of CNG1 mRNA in rat eye, lung, spleen, and brain. Polymerase chain reaction-based detection of the mRNA for CNG1 indicates that the channel is expressed in lower abundance in many other tissues, including thymus, skeletal muscle, heart, and parathyroid gland. The cellular distribution of CNG1 was further studied by in situ hybridization, which demonstrated expression of mRNA in lung, thymus, pineal gland, hippocampus, cerebellum, and cerebral cortex but not in heart or kidney.

Substance P receptor expression in intestinal epithelium inClostridium difficile toxin A enteritis in rats

1998 ◽  
Vol 275 (1) ◽  
pp. G68-G75 ◽  
Author(s):  
Charalabos Pothoulakis ◽  
Ignazio Castagliuolo ◽  
S. E. Leeman ◽  
Chi-Chung Wang ◽  
Hanzong Li ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Substance P ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Rat Intestine ◽  
Toxin A

We previously reported that the inflammatory effects of Clostridium difficile toxin A on rat intestine can be significantly inhibited with a specific neurokinin-1 receptor (NK-1R) antagonist. In this study we investigated the localization and expression of NK-1R mRNA and protein in rat intestine by in situ hybridization, Northern blot analysis, and immunohistochemistry, respectively, after exposure to toxin A. Northern blot analysis showed increased mucosal levels of NK-1R mRNA starting 30 min after toxin A administration. In situ hybridization showed that toxin A increased NK-1R mRNA expression in intestinal epithelial cells after 30, 120, and 180 min. In rats pretreated with the NK-1R antagonist CP-96345 the increase in NK-1R mRNA levels after exposure to toxin A was inhibited, indicating that NK-1R upregulation is substance P (SP) dependent. One hour after exposure to toxin A many of the intestinal epithelial cells showed staining for NK-1R compared with controls. Specific 125I-SP binding to purified epithelial cell membranes obtained from ileum exposed to toxin A for 15 min was increased twofold over control and persisted for 4 h. This report provides evidence that NK-1R expression is increased in the intestinal epithelium shortly after exposure to toxin A and may be important in toxin A-induced inflammation.

scholarly journals Expression and localization of mRNA for the 16 KD subunit of V-ATPase in the rat embryo.

1995 ◽  
Vol 43 (8) ◽  
pp. 761-769 ◽  
Author(s):  
M Numata ◽  
S Ohkuma ◽  
S Iseki
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mammalian Development ◽  
Mesenchymal Differentiation ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation

Vacuolar H(+)-ATPase (V-ATPase), an enzyme composed of multisubunits, is located in the membrane of intracellular organelles (e.g., lysosomes, and endosomes) and maintains the intraorganellar acidic pH by pumping protons across the membrane. Although there is growing evidence for some important role of V-ATPase in cell proliferation and differentiation, the functional significance of V-ATPase in vivo during mammalian development remains obscure. In the present study we investigated the expression and localization of mRNA for the 16 KD subunit of V-ATPase, an essential sector for enzymatic activity, in prenatal rat by Northern blot analysis and in situ hybridization with a specific oligonucleotide probe. With Northern blot analysis, consistent expression of the mRNA was observed in the embryos throughout the period examined (E14-E20). On in situ hybridization, mRNA signal was distributed with various intensities in both the epithelial and mesenchymal tissues at embryonic day 14 (E14). In E17 and E20 embryos, localization of strong signal became more restricted to distinct mesenchymal cells such as fibroblasts adjacent to the epithelia of skin, lung, and intestine, the cells of perichondrium, and myoblasts in the process of fusion. These results suggest that V-ATPase performs specific functions during the later stages of embryogenesis, especially at sites of mesenchymal differentiation and epithelium-mesenchyme interaction.

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

1988 ◽  
Vol 107 (2) ◽  
pp. 407-412 ◽  
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.

Thyrotropin receptor and leukocyte adhesion molecules in autoimmune thyroid disease: a study of their gene expression by Northern blot analysis and in situ hybridization

1994 ◽  
Vol 131 (5) ◽  
pp. 480-488 ◽  
Author(s):  
Frank Schuppert ◽  
Markus Reiser ◽  
Niels-E Heldin ◽  
Sülke Ede ◽  
Georg FW Scheumann ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Thyroid Disease ◽  
Autoimmune Thyroid Disease ◽  
Northern Blot Analysis ◽  
Leukocyte Adhesion ◽  
Immunological Parameters ◽  
Autoimmune Thyroid

Schuppert F, Reiser M, Heldin N-E, Ede S, Scheumann GFW, Dralle H, von zur Mühlen A. Thyrotropin receptor and leukocyte adhesion molecules in autoimmune thyroid disease: a study of their gene expression by Northern blot analysis and in situ hybridization. Eur J Endocrinol 1994;131:480–8. ISSN 0804–4643 In order to characterize the role of leukocyte-activating antigens and other immunological parameters in autoimmune thyroid disease, mRNA levels of intercellular adhesion molecule 1 (ICAM-1), endothelial leukocyte adhesion molecule 1 (ELAM-1, E-selectin), invariant chain (Ii) and the thymic hormone thymosin β4 (Tβ4) were investigated in 18 human thyroid glands, including eight Graves' thyroids, two Hashimoto's thyroids, two endemic goiters and six healthy controls. Northern blot analysis showed that in autoimmune thyroid disease, expression of ICAM-1 and Tβ4 was correlated to transcript levels of Ii, whereas in the healthy controls, expression of Tβ4, ICAM-1 and ELAM-1 was low or nearly absent. ELAM-1 and TSH receptor (TSH-R) expression, the latter serving as a thyroid specific marker, was increased in some diseased glands but showed no relation to the immunological parameters mentioned above. Localization of the specific mRNAs by in situ hybridization demonstrated a cell-specific expression of TSH-R (thyrocytes), ELAM-1 (vascular endothelial cells) and Tβ4 (cells of hematopoietic origin). In contrast, transcripts of Ii and ICAM-1 were found in thyrocytes, leukocytes and endothelial cells. Our results implicate a coordinate expression of ICAM-1, Tβ4 and Ii in autoimmune thyroid disease, yielding distinct cellular expression patterns. Differential expression of ICAM-1. Ii and the TSH-R in thyroid epithelial cells indicates active regulatory events within the thyrocyte. Frank Schuppert, Department of Clinical Endocrinology, Medizinische Hochschule Hannover, Konstanty-Gutschow-Strasse 8, D-30625 Hannover, Germany

Nerve Growth Factor Expression Correlates With Perineural Invasion and Pain in Human Pancreatic Cancer

1999 ◽  
Vol 17 (8) ◽  
pp. 2419-2419 ◽  
Author(s):  
Zhaowen Zhu ◽  
Helmut Friess ◽  
Fabio F. diMola ◽  
Arthur Zimmermann ◽  
Hans U. Graber ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
In Situ Hybridization ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Northern Blot ◽  
Blot Analysis ◽  
Perineural Invasion ◽  
Northern Blot Analysis ◽  
Human Pancreatic Cancer

PURPOSE: The reasons for the high frequency of perineural invasion and the presence of pain in pancreatic cancer are still not clear. Nerve growth factor (NGF) and its high-affinity receptor TrkA are involved in stimulating epithelial cancer cell growth and perineural invasion, as well as in pain generation in chronic benign disorders. PATIENTS AND METHODS: NGF and TrkA were examined by Northern blot analysis, in situ hybridization, and immunohistochemistry in 27 normal and 37 pancreatic cancer tissue samples. The molecular findings were correlated with the degree of perineural invasion, pain, and histopathologic tumor characteristics. RESULTS: Northern blot analysis indicated that NGF and TrkA mRNA levels were increased 2.7-fold and 5.6-fold, respectively (P < .05 and P < .05), in pancreatic cancer tissues compared with the normal pancreas tissue. As shown by in situ hybridization and immunohistochemistry, NGF was strongly present in the cytoplasm of pancreatic cancer cells. TrkA was intensely present in the perineurium of pancreatic nerves but not in the cancer cells. There was no difference in NGF and TrkA expression between early (stages I and II) and advanced (stage III) tumor stages and between well-/moderately differentiated (grades 1 and 2) and poorly differentiated (grade 3) tumors. However, tumors with high NGF/TrkA expression levels exhibited more frequent perineural invasion (P < .01). Furthermore, increased NGF/TrkA expression levels were associated with a higher degree of pain (P < .01). CONCLUSION: Enhanced expression of the NGF/TrkA system may influence perineural invasion and may contribute to the pain syndrome in human pancreatic cancer.

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