Abstract
Serial analysis of gene expression (SAGE) is a powerful genome-wide analytic tool to determine expression profiles. Since its description in 1995 by Victor Velculescu et al.1, SAGE has been widely used. Recently, the efficiency of the method has been emphasized as a means to identify novel transcripts or genes that are difficult to identify by conventional methods. SAGE is based on the principle that a 10-base pair (bp) cDNA fragment contains sufficient information to unambiguously identify a transcript, provided it is isolated from a defined position within this transcript. Concatenation of these sequence tags allows serial analysis of transcripts by sequencing multiple tags within a single clone. Extraction of sequence data by computer programs provides a list of sequence tags that reflect both qualitatively and quantitatively the gene expression profile. Several modifications to the initial protocol allowed to start from 1 μg total RNA (or 105 cells). In order to reduce the amount of input RNA, protocols including extra polymerase chain reaction (PCR) steps were designed. Linear amplification of the mRNA targets might have advantage over PCR by minimizing biases introduced by the amplification step; therefore we devised a SAGE protocol in which a loop of linear amplification of RNA has been included. Our approach, named “small amplified RNA-SAGE” (SAR-SAGE) included a T7 RNA polymerase promoter within an adapter derived from the standard SAGE linker. This allowed transcription of cDNA segments, extending from the last NlaIII site of transcripts to the polyA tail; these small amplified RNAs then serve as template in a classical (micro)SAGE procedure. As the cDNAs are immobilized on oligo(dT) magnetic beads, several rounds of transcription can be performed in succession with the same cDNA preparation, with the potential to increase further the yield in a linear way. Except for the transcription step itself, the present procedure does not introduce any extra enzymatic reaction in the classical SAGE protocol, it is expected to keep the representation biases associated with amplification as low as possible.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995) Serial analysis of gene expression. Science 270, 484–487.
Polyak, K. and Riggins, G. J. (2001) Gene discovery using the serial analysis of gene expression technique: implications for cancer research. J. Clin. Oncol. 19, 2948–2958.
Logan, M. (2002) SAGE profiling of the forelimb and hindlimb. Genome Biol. 3, REVIEWS1007.
Munasinghe, A., Patankar, S., Cook, B. P., et al. (2001) Serial analysis of gene expression (SAGE) in Plasmodium falciparum: application of the technique to A-T rich genomes. Mol. Biochem. Parasitol. 113, 23–34.
Chen, J., Sun, M., Lee, S., Zhou, G., Rowley, J. D., and Wang, S. M. (2002) Identifying novel transcripts and novel genes in the human genome by using novel SAGE tags. Proc. Natl. Acad. Sci. USA 99, 12257–12262.
Virlon, B., Cheval, L., Buhler, J. M., Billon, E., Doucet, A., and Elalouf, J. M. (1999) Serial microanalysis of renal transcriptomes. Proc. Natl. Acad. Sci. USA 96, 15286–15291.
St Croix, B., Rago, C., Velculescu, V., et al. (2000) Genes expressed in human tumor endothelium. Science 289, 1197–1202.
Ye, S. Q., Zhang, L. Q., Zheng, F., Virgil, D., and Kwiterovich, P. O. (2000) miniSAGE: gene expression profiling using serial analysis of gene expression from 1 microg total RNA. Anal. Biochem. 287, 144–152.
Peters, D. G., Kassam, A. B., Yonas, H., et al. (1999) Comprehensive transcript analysis in small quantities of mRNA by SAGE-lite. Nucleic Acids Res. 27, e39.
Datson, N. A., Perk-de Jong, J., van den Berg, M. P., de Kloet, E. R., and Vreugdenhil, E. (1999) MicroSAGE: a modified procedure for serial analysis of gene expression in limited amounts of tissue. Nucleic Acids Res. 27, 1300–1307.
Neilson, L., Andalibi, A., Kang, D., et al. (2000) Molecular phenotype of the human oocyte by PCR-SAGE. Genomics 63, 13–24.
Lee, S., Chen, J., Zhou, G., and Wang, S. M. (2001) Generation of high-quantity and quality tag/ditag cDNAs for SAGE analysis. Biotechniques 31, 348–354.
Vilain, C., Libert, F., Venet, D., Costagliola, S., and Vassart, G. (2003) Small Amplified RNA-SAGE: an alternative approach to study transcriptome from limiting amount of mRNA. Nucleic Acids Res. 31, e24.
Eberwine, J., Yeh, H., Miyashiro, K., et al. (1992) Analysis of gene expression in single live neurons. Proc. Natl. Acad. Sci. USA 89, 3010–3014.
Marble, H. A. and Davis, R. H. (1995) RNA transcription from immobilized DNA templates. Biotechnol. Prog. 11, 393–396.
Cheval, L., Virlon, B., and Elalouf, J. M. (2000) SADE: a microassay for serial analysis of gene expression, in Functional genomics: a practical approach (Hunt, S. P. and Liversey, J. P., ed.), Oxford University press, 139–163.
Blackshaw, S., Kim, J. B., St Croix, B., and Polyak, K. (2003) Serial Analysis of Gene Expression. In: Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R., et al., ed.) Massachusett’s General Hospital, Harvard Medical School, 25B.6.1–25B.6.29
Saha, S., Sparks, A. B., Rago, C., et al. (2002) Using the transcriptome to annotate the genome. Nat. Biotechnol. 20, 508–512.
Wang, E., Miller, L. D., Ohnmacht, G. A., Liu, E. T., and Marincola, F. M. (2000) High-fidelity mRNA amplification for gene profiling. Nat. Biotechnol. 18, 457–459.
Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., and Eberwine, J. H. (1990) Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA 87, 1663–1667.
Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001) Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res. 29, e29.
Pauws, E., Moreno, J. C., Tijssen, M., Baas, F., de Vijlder, J. J., and Ris-Stalpers, C. (2000) Serial analysis of gene expression as a tool to assess the human thyroid expression profile and to identify novel thyroidal genes. J. Clin. Endocrinol. Metab. 85, 1923–1927.
Iscove, N. N., Barbara, M., Gu, M., Gibson, M., Modi, C., and Winegarden, N. (2002) Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA. Nat. Biotechnol. 20, 940–943.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Vilain, C., Vassart, G. (2004). Small Amplified RNA-SAGE. In: Shimkets, R.A. (eds) Gene Expression Profiling. Methods in Molecular Biology, vol 258. Humana Press. https://doi.org/10.1385/1-59259-751-3:135
Download citation
DOI: https://doi.org/10.1385/1-59259-751-3:135
Publisher Name: Humana Press
Print ISBN: 978-1-58829-220-9
Online ISBN: 978-1-59259-751-2
eBook Packages: Springer Protocols