RNA profiling in peripheral blood cells by fluorescent differential display PCR.
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.
National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.
Martin
M
Steinau
Mangalathu S
MS
Rajeevan
D000818:Animals; D005453:Fluorescence; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000097:blood / Q000235:genetics*
D012313:RNA