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Methods Mol Biol. 2009;496:211-22. doi: 10.1007/978-1-59745-553-4_14.

RNA profiling in peripheral blood cells by fluorescent differential display PCR.

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1
National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.

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.

PMID:
18839113
DOI:
10.1007/978-1-59745-553-4_14
[Indexed for MEDLINE]
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