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

Abstract

The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semiquantitative analyses of changes in mRNA gene expression.

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

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

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

“…Unlike the high background produced by riboprobes 13 , oligonucleotide probes normally show a very low background. Thus, image can be easily and reasonably quantified using publicly-available NIH ImageJ software.…”

“…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.…”

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

“…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.…”

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

“…Unlike the high background produced by riboprobes 13 , oligonucleotide probes normally show a very low background. Thus, image can be easily and reasonably quantified using publicly-available NIH ImageJ software.…”

“…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.…”

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

“…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.…”

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

“…Unlike the high background produced by riboprobes 13 , oligonucleotide probes normally show a very low background. Thus, image can be easily and reasonably quantified using publicly-available NIH ImageJ software.…”

“…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.…”

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

“…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.…”