Small amplified RNA-SAGE: an alternative approach to study transcriptome from limiting amount of mRNA.
Serial analysis of gene expression (SAGE) is a widely used and powerful technique to characterize and compare transcriptomes. Although several modifications have been proposed to the initial protocol with the aim of reducing the amount of starting material, unless additional PCR steps are added, the technique is still limited by the need for at least 1 micro g of total RNA. As extra PCR amplification might introduce representation biases, current SAGE protocols are not fully suitable for the study of small, microdissected tissue samples. We propose here an alternative method involving the linear amplification of small mRNA fragments containing the SAGE tags. The procedure allows preparation of libraries of over 100 000 tags from as few as 2500 cells. A satisfactory correlation was observed between a microSAGE library made from 5 microg of total thyroid RNA, and a library prepared from 50 ng of the same RNA preparation according to the present protocol.
Vilain
Catheline
C
Institute of Interdisciplinary Research (IRIBHM), Université Libre de Bruxelles, 808 route de Lennik, B-1070 Bruxelles, Belgium. cavilain@ulb.ac.be
Libert
Frederick
F
Venet
David
D
Costagliola
Sabine
S
Vassart
Gilbert
G
D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013961:Thyroid Gland / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*
D012333:RNA, Messenger