Robust-LongSAGE (RL-SAGE): an improved LongSAGE method for high-throughput transcriptome analysis.
Serial analysis of gene expression (SAGE) is a powerful technique for large-scale transcriptome analysis in eukaryotes. However, technical difficulties in the SAGE library construction, such as low concatemer cloning efficiency, small concatemer size, and a high level of empty clones, has prohibited its widespread use as a routine technique for expression profiling in many laboratories. We recently improved the LongSAGE library construction method considerably and developed a modified version called Robust-LongSAGE, or RL-SAGE. In RL-SAGE, concatemer cloning efficiency and clone insert size were increased significantly. About 20 PCR reactions are sufficient to make a library with more than 150,000 clones. Using RL-SAGE, we have made 10 libraries of rice, maize, and the rice blast fungus Magnaporthe grisea.
Gowda
Malali
M
Ohio State University, Columbus, Ohio, USA.
Wang
Guo-Liang
GL
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; C049760:endodeoxyribonuclease MmeI; C048422:endodeoxyribonuclease NlaIII; D015252:Deoxyribonucleases, Type II Site-Specific