Open AccessAmplification of mRNA populations by a cDNA tag strategy
Author(s) -
Maria Sievertzon,
Charlotta Agaton,
Peter Nilsson,
Joakim Lundeberg
Publication year - 2004
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/04362st02
Subject(s) - complementary dna , biology , rapid amplification of cdna ends , microbiology and biotechnology , oligonucleotide , population , polymerase chain reaction , genetics , dna , computational biology , gene , molecular cloning , demography , sociology
Here we describe an amplification method for global transcript analysis. The strategy relies on amplification of cDNA tags (signature tags) achieved by random fragmentation of the cDNAs to short tags of similar length, isolation of the 3′ ends and then PCR amplification of the 3′-end signature tag population. This method minimizes biased amplification that may occur during parallel amplification of long and short templates. The amplified tags can be either cloned and sequenced or labeled and hybridized to DNA arrays to identify the expressed transcripts. To verify that the relative levels between transcripts in different mRNA/cDNA populations are maintained during the amplification protocol, we have used the Affymetrix oligonucleotide platform and real-time PCR.