RNA Profiling in Peripheral Blood Cells by Fluorescent Differential Display PCR

  • Martin Steinau
  • Mangalathu S. Rajeevan
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 496)

Abstract

The differential display-polymerase chain reaction (DD-PCR) technique is a unique, sequence independent tool for mRNA profiling and relative quantification. It is particularly suited for clinical samples yielding limited amounts of RNA. Unlike closed systems like microarray-based platforms, DD-PCR can be used to detect expression changes in known and novel transcripts, alternate splice products and to identify non-human transcripts. This chapter details fluorescent DD-PCR protocols that were optimized for peripheral blood mononuclear cells (PBMC). Subpopulations of mRNAs are reverse transcribed with two-base anchored oligo dT primers, amplified in combination with arbitrary primers and after gel separation visualized by fluorescent tags on the primers. Besides the DD-PCR itself, methods are described for subsequent extraction, amplification, and sequencing of DNA from bands of interest to identify the corresponding genes.

Key Words

Differential display PCR fluorescent detection blood RNA profiling PBMC 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Martin Steinau
    • 1
  • Mangalathu S. Rajeevan
    • 1
  1. 1.National Center for Zoonotic, Vector-Borne, and Enteric Diseases (NCZVED)Centers for Disease Control and Prevention (CDC)AtlantaUSA

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