Representational Difference Analysis


Representational difference analysis (RDA) is a highly sensitive technique for the identification of differences between complex deoxyribonucleic acid samples. RDA is based on polymerase chain reaction amplification in combination with multiple rounds of subtractive hybridization and can be used for the identification of unknown pathogens, genetic lesions in cancer and polymorphic markers linked to a trait without the use of a preexisting map, as well as for the identification of differentially expressed genes.

Keywords: RDA; representational difference analysis; amplicon; tester; driver; subtraction; subtractive hybridization; kinetic enrichment; differential expression; PCR; polymerase chain reaction; DNA; RNA; cDNA

Figure 1.

Schematic overview of representational difference analysis of complementary DNAs (cDNARDA) for the identification of differentially expressed genes. ds: double‐stranded.

Figure 2.

Effect of suppression of previously identified products in representational difference analysis (RDA). Ethidium bromide staining of an agarose gel loaded with driver (D) and tester (T) DNA amplicons and the difference products obtained after one and two rounds of subtractive hybridization respectively. The dramatic effect of RDA is clear: the difference product 2 consists mainly of two major products (indicated by triangles), derived from genes highly expressed in the cells from which the tester was derived only. A disadvantage of this very high level of purification of these two gene fragments is the fact that many other differentially expressed genes are underrepresented or absent in the difference product 2. However, cDNARDA offers the unique possibility to suppress the reappearance of previously identified products by adding those to the driver in a new RDA experiment. Difference products obtained after 1 (S1), 2 (S2) and 3 (S3) rounds of subtractive hybridization are shown; the two previously identified products are completely absent, and many additional differentially expressed genes can be identified this way (for details, see Groot and van Oost ).



Groot PC and van Oost BA (1998) Identification of fragments of human transcripts from a defined chromosomal region: representational difference analysis of somatic cell hybrids. Nucleic Acids Research 26(19): 4476–4481.

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

Li J, Yen C, Liaw D, et al. (1997) PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275: 1943–1947.

Lisitsyn N, Lisitsyn N and Wigler M (1993) Cloning the differences between two complex genomes. Science 259(5097): 946–951.

Lisitsyn NA, Lisitsina NM, Dalbagni G, et al. (1995) Comparative genomic analysis of tumors: detection of DNA losses and amplification. Proceedings of the National Academy of Sciences of the United States of America 92: 151–155.

Lisitsyn NA, Segre JA, Kusumi K, et al. (1994) Direct isolation of polymorphic markers linked to a trait by genetically directed representational difference analysis. Nature Genetics 6: 57–63.

Lowrey PL, Shimomura K, Antoch MP, et al. (2000) Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau. Science 288: 483–491.

Navin A, Prekeris R, Lisitsyn NA, et al. (1996) Mouse Y‐specific repeats isolated by whole chromosome representational difference analysis. Genomics 36: 349–353.

Schutte M, da Costa LT, Hahn SA, et al. (1995) Identification by representational difference analysis of a homozygous deletion in pancreatic carcinoma that lies within the BRCA2 region. Proceedings of the National Academy of Sciences of the United States of America 92: 5950–5954.

Further Reading

Everts RE, Versteeg SA, Renier C, et al. (2000) Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA). Mammalian Genome: Official Journal of the International Mammalian Genome Society 11: 741–747.

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

Michiels L, van Leuven F, van den Oord JJ, de Wolf‐Peeters C and Delabie J (1998) Representational difference analysis using minute quantities of DNA. Nucleic Acids Research 26: 3608–3610.

Muller K, Heller H and Doerfler W (2001) Foreign DNA integration: genome‐wide perturbations of methylation and transcription in the recipient genomes. Journal of Biological Chemistry 276: 14271–14278.

Sagerström CG, Sun BI and Sive HL (1997) Subtractive cloning: past, present, and future. Annual Review of Biochemistry 66: 751–783.

Simons JN, Pilot‐Matias TJ, Leary TP, et al. (1995) Identification of two flavivirus‐like genomes in the GB hepatitis agent. Proceedings of the National Academy of Sciences of the United States of America 92: 3401–3405.

Sornson MW and Rosenfeld MG (1999) Genetically directed representational difference analysis‐based positional cloning and Northern analysis of candidate genes. Methods in Enzymology 306: 67–89.

Toyota M, Canzian F, Ushijima T, et al. (1996) A rat genetic map constructed by representational difference analysis markers with suitability for large‐scale typing. Proceedings of the National Academy of Sciences of the United States of America 93: 3914–3919.

Wallrapp C and Gress TM (2001) Isolation of differentially expressed genes by representational difference analysis. Methods in Molecular Biology 175: 279–294.

Welford S, Gregg J, Chen E, et al. (1998) Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization. Nucleic Acids Research 26: 3059–3065.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Groot, Peter C, and van Oost, Bernard A(Sep 2005) Representational Difference Analysis. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0005374]