Analytical and Bioanalytical Chemistry

, Volume 400, Issue 10, pp 3383–3393 | Cite as

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

Original Paper
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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.

Keywords

Tissue lysis RNA purification Gene expression mapping Tissue section PCR 

Notes

Acknowledgments

We would like to thank Connie Rogers-Newcome at Adhesives Research for providing samples of ARseal 90697 sealing films.

Funding

This work was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

Supplementary material

216_2011_5062_MOESM1_ESM.pdf (140 kb)
ESM 1 Primer sequences used in this study, specific protocol for duplex mRNA mapping (ChargeSwitch buffer), specific protocol for triplex mRNA mapping (ChargeSwitch or custom GITC buffer), example of 2D-RTPCR without DNase treatment, fluorescent data and determination of Cts, binding of DNA to adhesives film (PDF 140 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Michael Armani
    • 1
    • 3
  • Michael A. Tangrea
    • 3
  • Benjamin Shapiro
    • 1
  • Michael R. Emmert-Buck
    • 3
  • Elisabeth Smela
    • 2
  1. 1.Fischell Department of Bio-EngineeringUniversity of MarylandCollege ParkUSA
  2. 2.Department of Mechanical EngineeringUniversity of MarylandCollege ParkUSA
  3. 3.Pathogenetics Unit, Laboratory of PathologyNational Cancer InstituteBethesdaUSA

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