Differential display

Differential display (also referred to as DDRT-PCR or DD-PCR) is a laboratory technique that allows a researcher to compare and identify changes in gene expression at the mRNA level between two or more eukaryotic cell samples.[1] It was the most commonly used method to compare expression profiles of two eukaryotic cell samples in the 1990s.[1] By 2000, differential display was superseded by DNA microarray approaches.[2]

In differential display, first all the RNA in each sample is reverse transcribed using a set of 3′ "anchored primers" (having a short sequence of deoxy-thymidine nucleotides at the end) to create a cDNA library for each sample, followed by PCR amplification using arbitrary 5′ primers together with anchored 3′ primers identical to those used to create the library; about thirty arbitrary primers can amplify almost all the mRNA. The resulting transcripts are then separated by electrophoresis and visualized, so that they can be compared.[1] The method was prone to error due to different mRNAs migrated into single bands, differences in less abundant mRNAs getting drowned by more abundant mRNAs,[1] sensitivity to small changes in cell culture conditions, and a tendency to amplify 3' fragments rather than full mRNAs, and the necessity to use about 300 primers to catch all the mRNA.[3]:316–317 The method was first published in Science in 1992.[1][4]

References

  1. 1 2 3 4 5 Peale FV, Jr; Gerritsen, ME (September 2001). "Gene profiling techniques and their application in angiogenesis and vascular development.". The Journal of pathology. 195 (1): 7–19. PMID 11568887.
  2. ↑ Pandey, A; Mann, M (15 June 2000). "Proteomics to study genes and genomes.". Nature. 405 (6788): 837–46. PMID 10866210.
  3. ↑ Reece, Richard J. (2004). Analysis of genes and genomes. Chichester: Wiley. ISBN 978-0-470-84380-2.
  4. ↑ Liang, P; Pardee, AB (14 August 1992). "Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.". Science (New York, N.Y.). 257 (5072): 967–71. PMID 1354393.
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