MicroSAGE: a modified procedure for serial analysis of gene expression in limited amounts of tissue.
Serial Analysis of Gene Expression (SAGE) is a powerful expression profiling method, allowing the analysis of the expression of thousands of transcripts simultaneously. A disadvantage of the method, however, is the relatively high amount of input RNA required. Consequently, SAGE cannot be used for the generation of expression profiles when RNA is limited, i.e. in small biological samples such as tissue biopsies or microdissected material. Here we describe a modification of SAGE, named microSAGE, which requires 500- to 5000-fold less starting material. Compared with SAGE, microSAGE is simplified due to incorporation of a 'single-tube' procedure for all steps from RNA isolation to tag release. Furthermore, a limited number of additional PCR cycles are performed. Using microSAGE gene expression profiles can be obtained from minute quantities of tissue such as a single hippocampal punch from a rat brain slice of 325 micrometers thickness, estimated to contain, at most, 10(5) cells. This method opens up a multitude of new possibilities for the application of SAGE, for example the characterization of expression profiles in tissue biopsies, tumor metastases or in other cases where tissue is scarce and the generation of region-specific expression profiles of complex heterogeneous tissues.
Datson
N A
NA
Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research, Leiden University, PO Box 9503, 2300 RA Leiden, The Netherlands. datson_n@lacdr.leidenuniv.nl
van der Perk-de Jong
J
J
van den Berg
M P
MP
de Kloet
E R
ER
Vreugdenhil
E
E
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D018891:Dentate Gyrus / Q000378:metabolism; D015870:Gene Expression; D005821:Genetic Techniques; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012680:Sensitivity and Specificity