Expression Studies

Abstract

Changes in gene expression underlie almost every biological process. Highly sensitive methods to monitor changes in expression of individual or complex groups of genes are now available.

Keywords: expression profile; quantitation; mRNA; transcript abundance; differential expression

Figure 1.

(a) S1 nuclease mapping and (b) primer extension. Asterisks represent (radioactive) labels to visualize DNA fragments. The differences in band intensities, represented by thin (sample 1) and thick lines (sample 2), are directly proportional to the amount of mRNA and thus give information on the level of gene expression.

Figure 2.

(a) 5′ rapid amplification of cDNA ends (RACE) and (b) 3′ RACE. mRNA is converted to cDNA and in the case of 5′ RACE, an adaptor is ligated to the 5′ end of the cDNA. Subsequently, the unknown 5′ or 3′ cDNA end is amplified by polymerase chain reaction (PCR) using a gene‐specific primer (GSP) complementary to a known part of the gene of interest in combination with an adaptor‐specific primer (ASP) (a) or a primer specific for the poly‐A tail (b).

Figure 3.

Suppressive subtractive hybridization. tester cDNA is divided into two equal portions and ligated to two different linkers (A, gray; B, black). Both tester portions are hybridized with excess driver cDNA resulting in tester/driver (or driver/driver) hybrids of sequences that tester and driver cDNA have in common. cDNA specific for the tester sample will not hybridize with driver cDNA. By combining both hybridized tester samples, and performing PCR using linker A‐ and B‐specific primers, tester‐specific cDNA can be amplified and cloned.

Figure 4.

Differential display. All mRNAs of a cell or tissue are reverse‐transcribed into cDNA using an anchored primer (5′ (T)11MN, wherein M = G, A or C and N is any base). The cDNA is divided into several pools and each pool is amplified using a pool‐specific arbitrarily chosen primer and an oligo(dT) primer in the presence of labeled dNTPs. This results in the amplification of a few hundred cDNAs per pool, which are size‐separated on polyacrylamide gels. Bands that are present in one sample but are not present or are weaker in the other represent upregulated genes (indicated by an arrow in sample 1) or, conversely, downregulated genes (indicated by an arrow in sample 2). By analyzing all pools of cDNAs, thousands of genes can be screened simultaneously.

Figure 5.

Serial analysis of gene expression (SAGE). In SAGE, short sequence tags (approximately 10 bp) are isolated from mRNA at a defined position, ligated to long multimers, cloned and sequenced. The frequency of each tag in the cloned multimers directly reflects transcript abundancy. In addition, the short tags are long enough to uniquely identify the corresponding transcript in database searches. Thus, SAGE results in an accurate picture of gene expression at both the qualitative and the quantitative levels.

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References

Alwine JC, Kemp DJ and Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl‐paper and hybridization with DNA probes. Proceedings of the National Academy of Sciences of the United States of America 74: 5350–5354.

Barnes WM (1994) PCR amplification of up to 35‐kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proceedings of the National Academy of Sciences of the United States of America 91: 2216–2220.

Berk AJ and Sharp PA (1977) Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease‐digested hybrids. Cell 12: 721–732.

Datson NA, van der Perk‐de Jong J, van den Berg MP, de Kloet ER and Vreugdenhil E (1999) MicroSAGE: a modified procedure for serial analysis of gene expression in limited amounts of tissue. Nucleic Acids Research 27: 1300–1307.

Diatchenko L, Lau YF, Campbell AP, et al. (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue‐specific cDNA probes and libraries. Proceedings of the National Academy of Sciences of the United States of America 93: 6025–6030.

Frohman MA, Dush MK and Martin GR (1988) Rapid production of full‐length cDNAs from rare transcripts: amplification using a single gene‐specific oligonucleotide primer. Proceedings of the National Academy of Sciences of the United States of America 85: 8998–9002.

Hubank M and Schatz DG (1994) Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Research 22: 5640–5648.

Kim S, Zeller K, Dang CV, Sandgren EP and Lee LA (2001) A strategy to identify differentially expressed genes using representational difference analysis and cDNA arrays. Analytical Biochemistry 288: 141–148.

Krug MS and Berger SL (1987) Determination of the molar concentration of messenger RNA. Methods Enzymology 152: 262–266.

Liang P and Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967–971.

Lisitsyn N, Lisitsyn N and Wigler M (1993) Cloning the differences between two complex genomes. Science 259: 946–951.

Livesey FJ and Hunt SP (1996) Identifying changes in gene expression in the nervous system: mRNA differential display. Trends in Neuroscience 19: 84–88.

Neilson L, Andalibi A, Kang D, et al. (2000) Molecular phenotype of the human oocyte by PCR‐SAGE. Genomics 63(1): 13–24.

Peters DG, Kassam AB, Yonas H, et al. (1999) Comprehensive transcript analysis in small quantities of mRNA by SAGE‐lite. Nucleic Acids Research 27(24): 39.

Sive HL and St John T (1988) A simple subtractive hybridization technique employing photoactivatable biotin and phenol extraction. Nucleic Acids Research 16: 10937.

Tsien RY and Miyawaki A (1998) Seeing the machinery of live cells. Science 280: 1954–1955.

Velculescu VE, Zhang L, Vogelstein B and Kinzler KW (1995) Serial analysis of gene expression. Science 270: 484–487.

Virlon B, Cheval L, Buhler JM, et al. (1999) Serial microanalysis of renal transcriptomes. Proceedings of the National Academy of Sciences of the United States of America 96(26): 15286–15291.

Ye SQ, Zhang LQ, Zheng F, Virgil D and Kwiterovich PO (2000) MiniSAGE: gene expression profiling using serial analysis of gene expression from 1 microg total RNA. Analytical Biochemistry 287(1): 144–152.

Further Reading

Leslie RA and Robertson HA (eds.) (2000) Differential Display: A Practical Approach. New York: Oxford University Press.

Lisitsyn NA (1995) Representational difference analysis: finding the differences between genomes. Trends in Genetics 11: 303–307.

Matz MV and Lukyanov A (1998) Different strategies of differential display: areas of application. Nucleic Acids Research 26: 5537–5543.

Naylor LH (1999) Reporter gene technology: the future looks bright. Biochemical Pharmacology 58: 749–757.

Velculescu VE, Vogelstein B and Kinzler KW (2000) Analysing uncharted transcriptomes with SAGE. Trends in Genetics 16: 423–425.

Web Links

NCBI Serial Analysis of Gene Expression Tag to Gene Mapping (SAGEmap) Mapping of SAGE tags to genes and vice versa and downloadable data from one hundred SAGE expression profiles of human tumors of the Cancer Genome Anatomy Project (CGAP) http://www.ncbi.nlm.nih.gov/SAGE/

SAGE: Serial Analysis of Gene Expression. An overview of a large number of SAGE publications, resources and helpful links. Furthermore, SAGE software and protocols can be requested http://www.sagenet.org.

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How to Cite close
Datson, Nicole, and Vreugdenhil, Erno(Jan 2006) Expression Studies. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0005673]