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

Ruifang Hua,Mugen Liu,Shanshan Yu,Haohong Li

doi:10.3389/fnins.2018.00266

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.

Highlights

In situ hybridization (ISH) is a powerful technique, which identifies the localization of specific genes to understand the structure and function of RNAs
Using transgenic reporter mice, we revealed the expression of the somatostatin (SST) gene in the adult mouse brain, which could contribute to understanding the heterogeneity in the neurogenic process
We introduce a simplified protocol to prepare DIG-labeled RNA probes based on polymerase chain reaction (PCR) amplification, and its various applications in neuroscience

Summary

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

Reviewed by: Wenzhi Sun, ShanghaiTech University, China Wei Shen, ShanghaiTech University, China Yang Xiang, UMass Memorial Medical Center, United States. In most cases, RNA probes are obtained by in vitro transcription from plasmids containing specific promoter elements and mRNA-specific cDNA. 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. The PCR-based method for non-radioactive RNA probes is a useful tool that can be substantially utilized in neuroscience studies

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION AND CONCLUSIONS