A PCR-Based Method for RNA Probes and Applications in Neuroscience

Abstract

In situ hybridization (ISH) is a powerful technique that is used to detect the localization of specific nucleic acid sequences for understanding the organization, regulation, and function of genes. However, in most cases, RNA probes are obtained by in vitro transcription from plasmids containing specific promoter elements and mRNA-specific cDNA. Probes originating from plasmid vectors are time-consuming and not suitable for the rapid gene mapping. Here, we introduce a simplified method to prepare digoxigenin (DIG)-labeled non-radioactive RNA probes based on polymerase chain reaction (PCR) amplification and applications in free-floating mouse brain sections. Employing a transgenic reporter line, we investigate the expression of the somatostatin (SST) mRNA in the adult mouse brain. The method can be applied to identify the colocalization of SST mRNA and proteins including corticotrophin-releasing hormone (CRH) and protein kinase C delta type (PKC-δ) using double immunofluorescence, which is useful for understanding the organization of complex brain nuclei. Moreover, the method can also be incorporated with retrograde tracing to visualize the functional connection in the neural circuitry. Briefly, the PCR-based method for non-radioactive RNA probes is a useful tool that can be substantially utilized in neuroscience studies.

Keywords: PCR; RNA probe; immuno-fluorescence; in situ hybridization; retrograde tracing; transgenic mice.

Figure 1

Preparation and visualization of SST probe in mouse brain sections. Two-step polymerase chain reaction (PCR) amplifications were performed and PCR products were examined by agarose gel electrophoresis. (A) Products of SST from the first PCR. (B) Products of SST containing the T7 promoter from the second PCR. (C) In vitro-transcribed RNA probe of SST. (D) Staining for SST mRNA expression revealed the effect of tissue permeability on signal intensity. The left images show treatment with 1 × PBST (1% Tween-20 in 0.01 M PBS) for 20 min at room temperature (RT); the middle images show treatment with 2 μg/ml proteinase K at RT; and the right images show treatment with 2 μg/ml proteinase K at 37°C. The upper images were captured using 10 × magnification (scale bar = 500 μm), and the bottom images were captured using 20 × magnification (scale bar = 100 μm). (E) Sections were stained with different concentrations of the SST probe at 0, 0.5, 2, and 4 μg/ml, respectively. Images were captured using 10 × magnification (scale bar = 200 μm). SST, somatostatin.

Figure 2

Detection of different mRNA species in mouse brain. (A) SST mRNA in the CeL. (B) NPSR1 mRNA in the BLA. (C) CCKBR mRNA in the CeL. The large images were captured with 10 × magnification (scale bar = 500 μm); the bottom-left images were captured with 20 × magnification (scale bar = 50 μm). NPSR1, neuropeptide S receptor 1; CCKBR, cholecystokinin B receptor; CeL, central lateral amygdala; BLA, basal lateral amygdala.

Figure 3

Application of ISH in transgenic reporter mice. SST expression in the CeL of the adult mouse brain that utilized (A) DAPI (blue), (B) immunofluorescence (IF) for protein(red), and (C) in situ hybridization for SST mRNA (black). (D) The co-localization of SST-IF, SST-ISH, and DAPI. Arrowheads indicate examples of neurons with colocalization; not all neurons with colocalization are indicated. (E) Quantification of the co-localization of SST-IF⁺ and SST-ISH⁺ (n = 3 mice). The data are presented as the mean ± S.E.M. DAPI, diamidino-phenylindole.

Figure 4

Combination of ISH with double immunofluorescence staining. Gene expression in the CeL that utilized (A) DAPI (blue), (B) in situ hybridization for SST mRNA (black), (C) immunofluorescence for PKC-δ protein (red), and (D) immunofluorescence for CRH protein (green). (E) The representative images for co-expression of SST, PKC-δ, CRH, and DAPI. The top images were obtained with 20 × magnification (scale bar = 50 μm), while the bottom images with 40 × magnification (scale bar = 15 μm). Arrowheads indicate examples of co-expressing neurons; not all co-expressing neurons are indicated. (F) Quantification of co-localization of SST⁺ and PKC-δ⁺. (G) Quantification of co-localization of PKC-δ⁺ and CRH⁺. (H) Quantification of co-localization of SST⁺ and CRH⁺. n = 3 mice. The data are presented as the mean ± S.E.M. PKC-δ, protein kinase C delta; CRH, adrenocorticotropic hormone.

Figure 5

ISH combined with retrograde tracer labeling to visualize the functional connection between CeL and SI. (A) Injection site of Alexa Fluor 555-CTB (red) in the SI. (B) The representative images of retrogradely labeled CeL neurons projecting to the SI, containing immunofluorescence staining for CTB (red), in situ hybridization for SST mRNA (black), immunofluorescence staining for PKC-δ protein (green), and DAPI (blue). The upper images were obtained with 20 × magnification (scale bar = 50 μm), while the bottom images with 40 × magnification (scale bar = 15 μm). Arrowheads indicate examples of neurons with colocalization; not all neurons with colocalization are indicated. (C) Quantification of co-localization of CTB⁺ and SST⁺. (D) Quantification of co-localization of CTB⁺ and PKC-δ⁺. n = 3 mice. The data are presented as the mean ± S.E.M. SI, substantia innominate.