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.

Molecular approaches underlying the oogenic cycle of the scleractinian coral, Acropora tenuis

“…Additional two pairs of PCR-based method RNA probes 113 of each genes were synthesised and tested to confirm the specificity of the RNA probes. Primary amplified products of PCR containing the specific DNA sequences were synthesised from cDNA.…”

MAFB modulates the maturation of lymphatic vascular networks in mice

“…The probe for Mafb was made as previously described 56 and spanned nucleotides 897 to 1873 of the mouse Mafb transcript ENSMUST00000099126.4. Sectional in situ hybridization (SISH) was performed on 7μm sections of paraformaldehyde‐fixed, paraffin‐embedded embryos.…”

A technical review and guide to RNA fluorescence in situ hybridization

“…Note, however, that glutaraldehyde is known to increase autofluorescence, at least with immunohistochemistry protocols. Whole mouse brains are often fixed in 4% PFA for up to 6 h at room temperature or overnight at 4 C, although fixation of brain tissue is recommended not to exceed 24 h (Kernohan & Bérubé, 2014;Kasai et al, 2016;Lanfranco et al, 2017;Hua et al, 2018).…”

“…The same treatment is also recommended for snail embryos as well as whole-mount planarian worms and is sometimes applied to fruit fly embryos, although several other permeabilization strategies including acetone are also frequently used for Drosophila (Paré et al, 2009;Pearson et al, 2009;Jackson, Herlitze & Hohagen, 2016;Hauptmann et al, 2016;Trcek et al, 2017). Some protocols call for brain sections to be treated with proteinase K, however, many protocols omit this step as permeability is less of an issue with sectioned material (Kasai et al, 2016;Hua et al, 2018). The proteinase K treatment will require careful optimization as too little digestion will prevent probe penetration whereas too much digestion will destroy the morphology of the tissue and lead to increased background (Tessmar-Raible et al, 2005;Bleckmann & Dresselhaus, 2016).…”