Comprehensive transcript analysis in small quantities of mRNA by SAGE-lite.
Serial analysis of gene expression (SAGE) is a powerful technique that can be used for global analysis of gene expression. Its chief advantage over other methods is that SAGE does not require prior knowledge of the genes of interest and provides quantitative and qualitative data of potentially every transcribed sequence in a particular tissue or cell type. Furthermore, SAGE can quantify low-abundance transcripts and reliably detect relatively small differences in transcript abundance between cell populations. However, SAGE demands high input levels of mRNA which are often unavailable, particularly when studying human disease. To overcome this limitation, we have developed a modification of SAGE that allows detailed global analysis of gene expression in extremely small quantities of tissue or cultured cells. We have called this approach 'SAGE-Lite'. This technique was used for the global analysis of transcription in samples of normal and pathological human cerebrovasculature to study the molecular pathology of intracranial aneurysms. These samples, which are obtained during operative surgical repair, are typically no bigger than 1 or 2 mm and yield <100 ng of total RNA. In addition, we show that SAGE-Lite allows simple and rapid isolation of long cDNAs from short (15 bp) SAGE sequence tags.
Peters
D G
DG
Department of Human Genetics, School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA. dpeters@helix.hgen.pitt.edu
Kassam
A B
AB
Yonas
H
H
O'Hare
E H
EH
Ferrell
R E
RE
Brufsky
A M
AM
D002460:Cell Line; D002941:Circle of Willis / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D002532:Intracranial Aneurysm / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013699:Temporal Arteries / Q000378:metabolism; D014158:Transcription, Genetic
D018076:DNA, Complementary