Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.     

Simultaneous detection of two messenger RNAs in the central nervous system: a simple two-step in situ hybridization procedure using a combination of radioactive and non-radioactive probes

We present here a method enabling the simultaneous detection of two messenger RNAs in tissue sections by use of a two-step in situ hybridization procedure. Tissue sections were hybridized with a radioactive probe and coated with emulsion. The emulsion was processed for development, fixed, and a second hybridization was performed through the emulsion with a biotinylated probe subsequently revealed with streptavidin-alkaline phosphatase. This procedure allows the detection of two mRNAs without loss of signal, removal of the emulsion, or spurious reaction. The simultaneous detection of oxytocin and vasopressin mRNAs in the hypothalamus, and of dopamine receptor and neuropeptide mRNAs in the striatum, demonstrated the efficiency of the procedure. Such a two-step procedure provides a simple and flexible way to make possible comparative analysis of the localization of two mRNAs within the same tissue section.     

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.     

Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.     

In situ hybridization using 32P labelled oligodeoxyribonucleotides for the cellular localisation of mRNA in neuronal and endocrine tissue. An analysis of procedural variables

Methodological variables for in situ hybridization using 32P labelled oligodeoxyribonucleotides (oligomers) have been examined. Four different oligomers directed against proglucagon messenger RNA (mRNA) and two different oligomers against prosomatostatin mRNA have been used. Specific hybridization was obtained in adult rat brain, stomach and pancreas and in neonatal rat ileum. Tissue was perfusion fixed with 4% paraformaldehyde 0.2% glutaraldehyde and hybridization was carried out in 50% formamide for 72 h at 42 degrees C. Using hybridization conditions of lower stringency (33% formamide) labelling was also obtained in guinea pig tissue. Other variables which affected hybridization signal intensity were the inclusion of a prehybridization dehydration stage, the probe concentration, the inclusion of ammonium acetate in the posthybridization dehydrating ethanols and in the autoradiographic emulsion, and the exposure time. The localisation of proglucagon mRNA in rat pancreas using a 20mer was used as a model tissue for testing these methodological variables and the results were found generally also to apply to the other probes and tissues tested. The methods described provide single cell resolution and show that 32P labelled oligomers may be used to localise neuropeptide and endocrine mRNAs in different types of tissue and in different mammalian species.     

Whole-cell hybridization of Methanosarcina cells with two new oligonucleotide probes.

Two new oligonucleotide probes targeting the 16S rRNA of the methanogenic genus Methanosarcina were developed. The probes have the following sequences (Escherichia coli numbering): probe SARCI551, 5'-GAC CCAATAATCACGATCAC-3', and probe SARCI645, 5'-TCCCGGTTCCAAGTCTGGC-3'. In situ hybridization with the fluorescently labelled probes required several modifications of standard procedures. Cells of Methanosarcina mazeii S-6 were found to lyse during the hybridization step if fixed in 3% formaldehyde and stored in 50% ethanol. Lysis was, however, not observed with cells fixed and stored in 1.6% formaldehyde-0.85% NaCl. Extensive autofluorescence of the cells was found upon hybridization in the presence of 5 mM EDTA, but successful hybridization could be obtained without addition of this compound. The mounting agent Citifluor AF1, often used in conjugation with the fluorochrome fluorescein, was found to wash the labelled probes out of the cells. Stable labelling could be obtained with rhodamine-labelled probes when the specimen was mounted in immersion oil, and high hybridization intensities of the Methanosarcina cells were found even in the presence of biomass from an anaerobic reactor. The inherent high autofluorescence of the biomass could be lowered by use of a highly specific narrow-band filter. The probes were found to be specific for Methanosarcina and useful for detection of this genus in samples from anaerobic reactors.     

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.     

Quantitation of GLUT1 and GLUT4 mRNA using a solution hybridization assay

The development of a solution hybridization assay for detecting GLUT1 and GLUT4 mRNA is described. The details of this assay are described in which copy RNA is used to quantitate messenger RNA in total RNA samples. This solution hybridization assay is highly specific and reproducible and is significantly more sensitive than Northern blotting. Since GLUT mRNAs can be quantitated in as little as 25 mg tissue, this technique is essential when the supply of tissue is limited. Furthermore, the elimination of gel-based separation techniques allows for mRNA quantitation in several hundred samples within two days following isolation of samples.     

Evidence of pre-prosomatostatin mRNA in human normal and tumoral anterior pituitary gland

Expression of the SRIH gene was investigated in six human normal anterior pituitaries, six GH-, three PRL-, three mixed GH/PRL-secreting and four nonsecreting adenomas. Total cellular RNA and poly(A+) mRNAs were analyzed by dot and Northern blot hybridization to a 3'-end labeled oligonucleotide probe specific for the human pre-proSRIH mRNA. A weak but detectable pre-proSRIH hybridization signal was present in human normal anterior pituitaries and in the four groups of adenomas. The size of this pre-proSRIH mRNA was indistinguishable from that found in our hypothalamic samples and close to that described in the literature. The wide variation of the signal intensity from one case to the other in each group of the different types of normal and tumoral antehypophyseal samples prevented establishment of any correlation between the level of pre-proSRIH mRNA and the nature of the pituitary tissue. The presence of SRIH mRNA in human normal and tumoral anterior pituitary tissues provides a sound basis to substantiate the hypothesis of a SRIH biosynthesis in the human anterior pituitary gland.     

In situ hybridization using 32P labelled oligodeoxyribonucleotides for the cellular localisation of mRNA in neuronal and endocrine tissue

Summary Methodological variables for in situ hybridization using ³²P labelled oligodeoxyribonucleotides (oligomers) have been examined. Four different oligomers directed against proglucagon messenger RNA (mRNA) and two different oligomers against prosomatostatin mRNA have been used. Specific hybridization was obtained in adult rat brain, stomach and pancreas and in neonatal rat ileum. Tissue was perfusion fixed with 4% paraformaldehyde 0.2% glutaraldehyde and hybridization was carried out in 50% formamide for 72 h at 42° C. Using hybridization conditions of lower stringency (33% formamide) labelling was also obtained in guinea pig tissue. Other variables which affected hybridization signal intensity were the inclusion of a prehybridization dehydration stage, the probe concentration, the inclusion of ammonium acetate in the posthybridization dehydrating ethanols and in the autoradiographic emulsion, and the exposure time. The localisation of proglucagon mRNA in rat pancreas using a 20mer was used as a model tissue for testing these methodological variables and the results were found generally also to apply to the other probes and tissues tested. The methods described provide single cell resolution and show that ³²P labelled oligomers may be used to localise neuropeptide and endocrine mRNAs in different types of tissue and in different mammalian species.     

Sequence complexity and tissue distribution of chick lens crystallin mRNAs

Using hybridization reactions with a cDNA copy, the complexity of polysomal polyadenylated mRNA from the day-old chick lens was found to correspond to 5800–7200 sequences of average size, arranged in three abundance classes. Experiments with heterologous cDNAs suggest on a qualitative basis that many of the sequences expressed in 8-day embryonic neural retina and pigmented epithelium mRNAs are also present in lens mRNA. A cDNA fraction complementary to the most abundant lens mRNAs, representing an approximate minimum of four sequences, was used to assay the dosage of putative crystallin sequences in these and other embryonic tissues. Neural retina and pigmented epithelium cytoplasmic mRNAs have low concentrations of these sequences, which appear to be absent from mRNA prepared from headless bodies and muscle.     

Accumulation of creatine kinase mRNA during myogenesis: molecular cloning of a B-creatine kinase cDNA

Cytosolic creatine kinase isoenzymes MM, MB, and BB are assembled from M or B subunits which occur in different relative amounts in specific tissues. The accumulation of mRNAs encoding the M and B subunits was measured during myogenesis in culture. The relative concentration of the two mRNAs was determined by hybridization with a M-CK cDNA probe isolated previously and a B-CK cDNA probe, the cloning and characterization of which is reported here. The B-CK cDNA hybridizes specifically to a 1.6-kb mRNA found in brain and gizzard but not in adult skeletal muscle tissue. The M-CK cDNA hybridizes to a smaller mRNA 1.4-kb long which is specific to skeletal muscle. In culture, the B-CK mRNA is transiently induced and then declines to a low but detectable level.     

Methacarn fixation--effects of tissue processing and storage conditions on detection of mRNAs and proteins in paraffin-embedded tissues

In this study, we examined suitable conditions for tissue fixation with methacarn and ethanol dehydration and storage of paraffin-embedded tissues (PETs) on gene expression analysis. With fixation and dehydration of rat liver tissues for up to 16 h (overnight) and 1 week, respectively, at 4 degrees C, integrity of extracted total RNAs and polypeptides did not vary, the former integrity being constantly lower than that with unfixed frozen tissue, while protein yield was slightly reduced with increasing dehydration. Retained expression levels of mRNAs and proteins were mostly unaffected by the period of fixation but slightly fluctuated with the length of dehydration. When PETs were stored for up to 12 months, integrity of both total RNAs and polypeptides was retained at 4 degrees C but reduced at room temperature. Reduced expression levels of mRNAs and proteins were also noted by storage at room temperature after 12 and 3 months, respectively. However, neither tissue processing nor storage affected variability in either mRNA or protein levels among samples. Thus, the results suggest that, for gene expression analysis, tissues can be fixed with methacarn and dehydrated for at least 1 day and 1 week, respectively, and PETs can be stored for at least 12 months, but a temperature of 4 degrees C is preferable.     

Tissue and development specific regulation of a complex family of rat insulin-like growth factor I messenger ribonucleic acids

To obtain information about the functional significance of the structural heterogeneity that has been described for rat insulin-like growth factor I (IGF-I) cDNAs, we hybridized polyadenylated RNAs from rat tissues at different developmental stages with probes specific for two variant 5'-sequences (designated here as type 1 and type 2), with a probe specific for IB type E domain coding sequences and with a probe for E domain sequences common to IA and IB type IGF-I cDNAs. Northern blot analyses revealed that previously reported rat liver IGF-I mRNAs of estimated size 7.5-7.0, 1.9-1.5, and 1.2-0.9 kilobases each are comprised of multiple closely migrating IGF-I mRNA species containing either of two 5'-sequences and either IA or IB type E domain coding sequences. In liver, each of these detected IGF-I mRNA species showed postnatal increases in abundance. The mRNAs detected with the probe for type 2 5'-sequences were detected exclusively in postnatal liver and also showed a different pattern of postnatal increase in abundance than other IGF-I mRNA types. IGF-I mRNAs detected with the probe for IB type E domain coding sequences likewise were highly liver specific and were undetectable or barely detectable in other fetal or adult rat tissues. In contrast, IGF-I mRNAs that hybridized with probes for type 1 5'-sequences or for E domain coding sequences common to IA and IB type IGF-I mRNAs were detected in all fetal and adult rat tissues tested. These findings suggest development and tissue specific regulation of the expression of different rat IGF-I mRNA types, and also suggest a possible role of different precursor sequences encoded by the various mRNAs in targeting of IGF-I to a local site of action.     

Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization

Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.     

A general method for detection and characterization of an mRNA using an oligonucleotide probe

A general method for the detection and characterization of an mRNA using an oligodeoxynucleotide probe is described. The results presented indicate that a G-dT or a dG-U base pair within a short DNA-RNA hybrid does not significantly reduce the stability of the hybrid. On this basis, we propose that 11 amino acids, including Trp and Met, can be used in selecting a peptide sequence suitable for use in designing an oligodeoxynucleotide probe complementary to a given mRNA. To test this hypothesis, we have synthesized an oligodeoxynucleotide (oligo II) complementary to the region of gastrin mRNA coding for Trp-Met-Asp-Phe and have compared its effectiveness as a hybridization probe and as a primer for the synthesis of gastrin-specific cDNA with another oligonucleotide (oligo I) complementary to the region of gastrin mRNA coding for Trp-Met-Glu-Glu. Unlike oligo I, oligo II functions as a primer in specific cDNA synthesis only when the mRNA is denatured prior to initiation of synthesis. Similarly, oligo II can be used as a specific hybridization probe for gastrin mRNA only when the RNA is denatured and partially cleaved with NaOH before hybridization. A simple procedure for denaturing gastrin mRNA to ensure efficient interaction with oligodeoxynucleotide probes is described. This procedure should be useful in studies with other oligonucleotides and mRNAs as well.