Amplification of mRNA populations by a cDNA tag strategy.
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.
Royal Institute of Technology, Stockholm, Sweden.
Maria
M
Sievertzon
Charlotta
C
Agaton
Peter
P
Nilsson
Joakim
J
Lundeberg
D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D018076:DNA, Complementary; D012333:RNA, Messenger