Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

Carter, Bradley S.; Fletcher, Jonathan S.; Thompson, Robert C.

doi:10.1016/j.ymeth.2010.08.001

Cited by 16 publications

(17 citation statements)

References 46 publications

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“…Probes were purified using BioRad P-6 columns; the effluent was measured with a scintillation counter and then diluted with hybridization solution (~2 × 10 6 cpms of probe in 40 μl hybridization buffer per slide). All remaining tissue processing steps were performed according to published lab protocols (Carter et al, 2010). Probe hybridization specificity was defined in control experiments by sense probes which failed to yield autoradiographic signals above background.…”

Section: Methodsmentioning

confidence: 99%

Acute and chronic glucocorticoid treatments regulate astrocyte-enriched mRNAs in multiple brain regions in vivo

Carter¹,

Hamilton²,

Thompson³

2013

Front. Neurosci.

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Previous studies have primarily interpreted gene expression regulation by glucocorticoids in the brain in terms of impact on neurons; however, less is known about the corresponding impact of glucocorticoids on glia and specifically astrocytes in vivo. Recent microarray experiments have identified glucocorticoid-sensitive mRNAs in primary astrocyte cell culture, including a number of mRNAs that have reported astrocyte-enriched expression patterns relative to other brain cell types. Here, we have tested whether elevations of glucocorticoids regulate a subset of these mRNAs in vivo following acute and chronic corticosterone exposure in adult mice. Acute corticosterone exposure was achieved by a single injection of 10 mg/kg corticosterone, and tissue samples were harvested 2 h post-injection. Chronic corticosterone exposure was achieved by administering 10 mg/mL corticosterone via drinking water for 2 weeks. Gene expression was then assessed in two brain regions associated with glucocorticoid action (prefrontal cortex and hippocampus) by qPCR and by in situ hybridization. The majority of measured mRNAs regulated by glucocorticoids in astrocytes in vitro were similarly regulated by acute and/or chronic glucocorticoid exposure in vivo. In addition, the expression levels for mRNAs regulated in at least one corticosterone exposure condition (acute/chronic) demonstrated moderate positive correlation between the two conditions by brain region. In situ hybridization analyses suggest that select mRNAs are regulated by chronic corticosterone exposure specifically in astroctyes based on (1) similar general expression patterns between corticosterone-treated and vehicle-treated animals and (2) similar expression patterns to the pan-astrocyte marker Aldh1l1. Our findings demonstrate that glucocorticoids regulate astrocyte-enriched mRNAs in vivo and suggest that glucocorticoids regulate gene expression in the brain in a cell type-dependent fashion.

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Section: Methodsmentioning

confidence: 99%

Acute and chronic glucocorticoid treatments regulate astrocyte-enriched mRNAs in multiple brain regions in vivo

Carter¹,

Hamilton²,

Thompson³

2013

Front. Neurosci.

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“…Applications are very diverse including the analysis of gene expression; the study of molecular mechanisms implicated in certain diseases; or the detection of pathogenic agents [15][16][17]. Those application for the detection of fungus or bacteria are usually based on fluorescence (FISH), by using fluorochromes as reporter molecules [15].…”

Section: Resultsmentioning

confidence: 99%

A New Colorimetric Peptide Nucleic Acid-Based Assay for the Specific Detection of Bacteria

et al. 2014

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Aim: Developments on synthetic molecules, such as peptide nucleic acid (PNA), make FISH procedures more robust for microbial identification. Fluorochromes use might hinder a broader implementation of PNA-FISH, but colorimetric applications are inexistent so far. Methods: A biotin-labeled eubacteria probe was used to develop a colorimetric PNA-in situ hybridization (ISH) assay. An enzymatic-conjugate, targeting biotin, was introduced. The procedure was optimized and evaluated regarding sensitivity, specificity and detection limit. Results: Results have shown strong ISH signals. The method was specific, but permeabilization problems were observed for Gram-positive bacteria. Detection limit was 5 × 10 7 CFU/ml, limiting current applications to pre-enriched samples. Conclusion: The PNA-ISH procedure described here is a simple alternative to other detection methods, and is also the base for the development of other PNA colorimetric systems.

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“…However, enzyme activity can be quickly distinguished by placing the tissue in the dry ice/alcohol solution 5,12 . Although quick preservation is critical for hybridization using a riboprobe 13 , little is known about experimental conditions for oligonucleotide probes. In this study using oligonucleotide probes, we found that there was no difference between the paraformaldehyde-prefixed and fresh brains quickly preserved in the ice/alcohol solution.…”

Section: Discussionmentioning

confidence: 99%

Rapid <em>In Situ</em> Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

Shokry

Callanan

Sousa

et al. 2015

JoVE

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3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

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Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

Cited by 16 publications

References 46 publications

Acute and chronic glucocorticoid treatments regulate astrocyte-enriched mRNAs in multiple brain regions in vivo

Acute and chronic glucocorticoid treatments regulate astrocyte-enriched mRNAs in multiple brain regions in vivo

A New Colorimetric Peptide Nucleic Acid-Based Assay for the Specific Detection of Bacteria

Rapid <em>In Situ</em> Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

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