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A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.

Stylianopoulou E, Lykidis D, Ypsilantis P, Simopoulos C, Skavdis G, Grigoriou M - PLoS ONE (2012)

Bottom Line: In addition, a 50-60% shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method.Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFA-fixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method.Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highly sensitive, and especially suitable to implement in the study of genes expressed at low levels and/or in sparse cells within a structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece.

ABSTRACT

Background: Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels.

Methodology/principal findings: A stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50-60% shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFA-fixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method.

Conclusions/significance: Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highly sensitive, and especially suitable to implement in the study of genes expressed at low levels and/or in sparse cells within a structure.

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A rapid NoRISH protocol which allows clear detection of the cellular localization of the signal.In situ hybridization on sections of E13.5 mouse embryos fixed with Z7 for 1 h (A, C, E, G, I, K) or PFA for 24 h (B, D, F, H, J, L) and hybridized with antisense RNA probes against three different genes. ncapg (A–D), Lhx7 (E–H), ret (I–L). Detection time: A, C: 10 h - B, D: 36 h - E, G: 3 h - F, H: 6 h - I, K: 3 h - J, L: 10 h. cx: cortex, drg: dorsal root ganglion, mge: medial ganglionic eminence, mes: mesencephalon, lge: lateral ganglionic eminence, oe: oral epithelium, om: oral mesenchyme, st: stomach. Scale bar: 100 µm.
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pone-0033898-g003: A rapid NoRISH protocol which allows clear detection of the cellular localization of the signal.In situ hybridization on sections of E13.5 mouse embryos fixed with Z7 for 1 h (A, C, E, G, I, K) or PFA for 24 h (B, D, F, H, J, L) and hybridized with antisense RNA probes against three different genes. ncapg (A–D), Lhx7 (E–H), ret (I–L). Detection time: A, C: 10 h - B, D: 36 h - E, G: 3 h - F, H: 6 h - I, K: 3 h - J, L: 10 h. cx: cortex, drg: dorsal root ganglion, mge: medial ganglionic eminence, mes: mesencephalon, lge: lateral ganglionic eminence, oe: oral epithelium, om: oral mesenchyme, st: stomach. Scale bar: 100 µm.

Mentions: The results of the in situ hybridization experiments performed with the antisense probes against ncapg, Lhx7 and ret are presented in Table 2, Figure 3, Figure 4 and Figure S1. At E13.5 Lhx7 expression appeared in cell populations of the developing telencephalon and oral mesenchyme. On the other hand, at this stage, ret expression was restricted in subsets of neuronal cells in the developing brain (not shown), the dorsal root ganglia and the gut. Finally, at this stage ncapg was expressed in the ventricular zone of the developing brain. As in the case of Lhx6, the signal of Lhx7, ret and ncapg appeared earlier on the Z7-fixed tissues (Table 3). Moreover, at any specific time point during the linear phase of the colorimetric detection period, as well as at the end of the detection, the signal on the Z7-fixed sections was always stronger than that of the PFA-fixed tissues (Fig. 3). Compared to PFA-fixed tissues the detection time for Z7-fixed tissues was reduced by approximately 60% (Table 2). For example, the time required to detect the ncapg probe on the sections of the Z7-fixed embryos (between 10 and 12 h; Fig. 3 A, C) was considerably less than the time required for the detection of the same probe on the sections of the PFA-fixed embryos (between 36 and 48 h; Fig. 3 B, D). Similar results were obtained for Lhx7 (3 h detection time for the sections of the Z7-fixed embryos, 6 h for the sections of the PFA-fixed embryos - compare Fig. 3E, G with 3F, H) and ret (3 h detection time for the sections of the Z7-fixed embryos, 10 h for the sections of the PFA-fixed embryos - compare Fig. 3 I, K with 3 J, L). Control experiments with the sense probes against ncapg, Lhx7 and ret showed that the improvement of the sensitivity of the method did not compromise its specificity.


A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.

Stylianopoulou E, Lykidis D, Ypsilantis P, Simopoulos C, Skavdis G, Grigoriou M - PLoS ONE (2012)

A rapid NoRISH protocol which allows clear detection of the cellular localization of the signal.In situ hybridization on sections of E13.5 mouse embryos fixed with Z7 for 1 h (A, C, E, G, I, K) or PFA for 24 h (B, D, F, H, J, L) and hybridized with antisense RNA probes against three different genes. ncapg (A–D), Lhx7 (E–H), ret (I–L). Detection time: A, C: 10 h - B, D: 36 h - E, G: 3 h - F, H: 6 h - I, K: 3 h - J, L: 10 h. cx: cortex, drg: dorsal root ganglion, mge: medial ganglionic eminence, mes: mesencephalon, lge: lateral ganglionic eminence, oe: oral epithelium, om: oral mesenchyme, st: stomach. Scale bar: 100 µm.
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Related In: Results  -  Collection

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pone-0033898-g003: A rapid NoRISH protocol which allows clear detection of the cellular localization of the signal.In situ hybridization on sections of E13.5 mouse embryos fixed with Z7 for 1 h (A, C, E, G, I, K) or PFA for 24 h (B, D, F, H, J, L) and hybridized with antisense RNA probes against three different genes. ncapg (A–D), Lhx7 (E–H), ret (I–L). Detection time: A, C: 10 h - B, D: 36 h - E, G: 3 h - F, H: 6 h - I, K: 3 h - J, L: 10 h. cx: cortex, drg: dorsal root ganglion, mge: medial ganglionic eminence, mes: mesencephalon, lge: lateral ganglionic eminence, oe: oral epithelium, om: oral mesenchyme, st: stomach. Scale bar: 100 µm.
Mentions: The results of the in situ hybridization experiments performed with the antisense probes against ncapg, Lhx7 and ret are presented in Table 2, Figure 3, Figure 4 and Figure S1. At E13.5 Lhx7 expression appeared in cell populations of the developing telencephalon and oral mesenchyme. On the other hand, at this stage, ret expression was restricted in subsets of neuronal cells in the developing brain (not shown), the dorsal root ganglia and the gut. Finally, at this stage ncapg was expressed in the ventricular zone of the developing brain. As in the case of Lhx6, the signal of Lhx7, ret and ncapg appeared earlier on the Z7-fixed tissues (Table 3). Moreover, at any specific time point during the linear phase of the colorimetric detection period, as well as at the end of the detection, the signal on the Z7-fixed sections was always stronger than that of the PFA-fixed tissues (Fig. 3). Compared to PFA-fixed tissues the detection time for Z7-fixed tissues was reduced by approximately 60% (Table 2). For example, the time required to detect the ncapg probe on the sections of the Z7-fixed embryos (between 10 and 12 h; Fig. 3 A, C) was considerably less than the time required for the detection of the same probe on the sections of the PFA-fixed embryos (between 36 and 48 h; Fig. 3 B, D). Similar results were obtained for Lhx7 (3 h detection time for the sections of the Z7-fixed embryos, 6 h for the sections of the PFA-fixed embryos - compare Fig. 3E, G with 3F, H) and ret (3 h detection time for the sections of the Z7-fixed embryos, 10 h for the sections of the PFA-fixed embryos - compare Fig. 3 I, K with 3 J, L). Control experiments with the sense probes against ncapg, Lhx7 and ret showed that the improvement of the sensitivity of the method did not compromise its specificity.

Bottom Line: In addition, a 50-60% shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method.Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFA-fixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method.Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highly sensitive, and especially suitable to implement in the study of genes expressed at low levels and/or in sparse cells within a structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece.

ABSTRACT

Background: Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels.

Methodology/principal findings: A stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50-60% shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFA-fixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method.

Conclusions/significance: Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highly sensitive, and especially suitable to implement in the study of genes expressed at low levels and/or in sparse cells within a structure.

Show MeSH
Related in: MedlinePlus