miniSAGE: gene expression profiling using serial analysis of gene expression from 1 microg total RNA.
The use of serial analysis of gene expression (SAGE) to determine gene expression profiles is increasing because the technique can provide absolute transcript numbers in a digital format and identify new genes. We developed a miniSAGE technique, which uses only 1 microg total RNA and reduces the amount of the starting material by 250- to 500-fold. Unlike the other modified SAGE methods, the miniSAGE technique does not require the additional PCR amplifications. The additional PCR amplifications potentially introduce bias and compromise the quantitative aspects of the SAGE method. Three key modifications in the miniSAGE technique are: (i) using the phase lock gel (PLG, Eppendorf) to increase the recovery and the purity of DNA material after each phenol extraction step; (ii) reducing the amount of linkers in the ligation, thereby minimizing their interference with SAGE ditag amplification and increasing the SAGE ditag yield; and (iii) employing the mRNA capture kit (Boehringer Mannheim) to allow the first five steps: mRNA isolation, cDNA synthesis, enzyme cleavage of cDNA, binding of the cleaved biotin-cDNA to the streptavidin-magnetic beads, ligating linkers to the bound cDNA, and the release of cDNA tags to occur within one tube to significantly reduce the loss of material between successive steps. Two fibroblast SAGE libraries have been successfully prepared. The preliminary analysis of 3838 tags from one library demonstrated a typical fibroblast gene expression pattern. This miniSAGE technique will permit a broader application of SAGE.
Ye
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Lipid Research Atherosclerosis Division, Department of Pediatrics, CMSC 604, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, Maryland 21287, USA. syq@mail.jhmi.edu
Zhang
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LQ
Zheng
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Virgil
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D
Kwiterovich
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PO
D001706:Biopsy; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods*; D005821:Genetic Techniques; D006801:Humans; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012867:Skin / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA