Improved NlaIII digestion of PAGE-purified 102 bp ditags by addition of a single purification step in both the SAGE and microSAGE protocols.
Despite the success of microarray technologies, serial analysis of gene expression (SAGE) still remains the only technique that allows an accurate quantitative and qualitative analysis of cell transcription in a variety of physiological and pathological conditions. Nevertheless, the efficiency of SAGE is limited by the numerous gel purification steps required and these increase the possibility of contamination and reduce or inhibit the activity of the enzymes used in the protocol. In order to eliminate this problem, we have modified the original protocol by adding a single purification step before NLA:III digestion of the ditags. This allows us to increase the yield of digested ditags without reducing the amount of DNA or affecting the subsequent concatemerization.
Angelastro
J M
JM
Department of Pathology and Taub Center for Alzheimer's Disease Research and Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA. jma14@columbia.edu
Klimaschewski
L P
LP
Vitolo
O V
OV
D000818:Animals; D002498:Centrifugation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling / Q000379:methods*; D016716:PC12 Cells; D051381:Rats; D011933:Reagent Kits, Diagnostic; D012822:Silicon Dioxide
D011933:Reagent Kits, Diagnostic; D012822:Silicon Dioxide; C048422:endodeoxyribonuclease NlaIII; D015252:Deoxyribonucleases, Type II Site-Specific