Generation of high-quantity and quality tag/ditag cDNAs for SAGE analysis.
The serial analysis of gene expression (SAGE) technique is an important tool for genome-wide gene expression analysis. However, the requirement of a large amount of mRNA for the analysis and the difficulties in generating high-quality tag and ditag fragments for the construction of a SAGE library often interfere with the successful performance of the SAGE technique. We developed two procedures to solve these issues: (i) introducing low-cycle PCR amplification of the 3' cDNA before the BsmFI digestion of the 3' cDNAs and (ii) gel purifying the BsmFI-released tag fragments before ditag formation. These modifications provide a large quantity of initial 3' cDNAs and high-quality tags and ditags for the construction of SAGE libraries.
Lee
S
S
University of Chicago Medical Center, 5841 S. Maryland MC2115, Chicago, IL 60637, USA.
Chen
J
J
Zhou
G
G
Wang
S M
SM
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger; C048422:endodeoxyribonuclease NlaIII; D015252:Deoxyribonucleases, Type II Site-Specific