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.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Steinau, M., Rajeevan, M.S. (2009). RNA Profiling in Peripheral Blood Cells by Fluorescent Differential Display PCR. In: Bugert, P. (eds) DNA and RNA Profiling in Human Blood. METHODS IN MOLECULAR BIOLOGY™, vol 496. Humana Press. https://doi.org/10.1007/978-1-59745-553-4_14
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DOI: https://doi.org/10.1007/978-1-59745-553-4_14
Publisher Name: Humana Press
Print ISBN: 978-1-934115-93-0
Online ISBN: 978-1-59745-553-4
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