Quantifying mRNA levels across tissue sections with 2D-RT-qPCR.

ABSTRACT: Measurement of mRNA levels across tissue samples facilitates an understanding of how genes function and what their roles are in disease. Quantifying low-abundance mRNA requires a workflow that preserves spatial information, isolates RNA, and performs reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). This is complex because these steps are typically performed in three separate platforms. In the present study, we describe two-dimensional RT-qPCR (2D-RT-qPCR), a method that quantifies RNA across tissues sections in a single integrated platform. The method uses the grid format of a multi-well plate to macrodissect tissue sections and preserve the spatial location of the RNA; this also eliminates the need for physical homogenization of the tissue. A new lysis and nucleic acid purification protocol is performed in the same multi-well plate, followed by RT-qPCR. The feasibility 2D-RT-qPCR was demonstrated on a variety of tissue types. Potential applications of the technology as a high-throughput tissue analysis platform are discussed.

SUBMITTER: Armani M 

PROVIDER: S-EPMC7375691 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature