title: 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.
authors.affiliation: Fischell Department of Bio-Engineering, University of Maryland, College Park, MD 20742, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Armani
authors.forename: Michael A
authors.initials: MA
authors.lastname: Tangrea
authors.forename: Benjamin
authors.initials: B
authors.lastname: Shapiro
authors.forename: Michael R
authors.initials: MR
authors.lastname: Emmert-Buck
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Smela
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D014018:Tissue Distribution
chemical_list: D012333:RNA, Messenger