RESUMEN
Characterizing the in vivo biodistribution pattern and relative expression levels of oligonucleotide-based molecules such as mRNA, miRNA, siRNA, and anti-miRNAs in animal models, could be a helpful first-step in the successful development of therapeutic oligonucleotides. Here we describe a simple procedure called "Whole-Body Scanning PCR" (WBS-PCR), which combines the power of PCR with that of imaging. WBS-PCR relies on 384 well-defined extractions across a mouse whole-body section followed by a single dilution step which renders the lysates compatible with various qPCR-based assays. The in vivo biodistribution maps are generated by deconvoluting the qPCR data and converting it into a TissueView compatible image file which can be overlaid with an image of the whole-body section used for extractions. WBS-PCR is a flexible platform that can be adapted to other detection systems and thereby further expand the use of this technology.
Asunto(s)
Expresión Génica , Imagen Molecular/métodos , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Imagen de Cuerpo Entero/métodos , Animales , Crioultramicrotomía/métodos , Ratones , MicroARNs/genética , Modelos Animales , Oligonucleótidos/administración & dosificación , ARN Mensajero/genética , ARN Ribosómico 18S/genética , ARN Interferente Pequeño/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Roedores , Distribución TisularRESUMEN
Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.
