Methods (molecular, IHC, frozen)

Topic Completed: 1 July 2010

Minor changes: 8 July 2020

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PubMed Search: Microarray[TI] molecular[TI] free full text[sb]

Rodney E. Shackelford, D.O., Ph.D.
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Cite this page: Shackelford RE. Microarray-introduction. website. Accessed January 18th, 2021.
Definition / general
  • Microarray consists of a solid support onto which DNA probes of known sequence are fixed in an orderly arrangement
  • The slide is hybridized under high stringency conditions with labeled nucleic acid targets (often mRNA or cDNA), extensively washed and the relative amount of probe bound target sequence is measured via label detection
  • Microarray allows the massive, parallel, semiquantitative analysis of the relative gene (nucleic acid) expression levels between two different cell populations, typically comparing "control" to "treated" cell populations or primary cell populations to neoplastic ones of the same or similar lineage
  • The number of different gene / nucleic acid sequences that can be simultaneously assayed on one DNA chip can range from ten to one million
  • Microarray technology, with its ability to simultaneously examine the expression of many different genes, has changed our approach to research; instead of asking "does drug X induce gene Y?", it is possible to ask "what set of genes are induced by drug X?"
  • Earliest form of microarray is the Southern blot, developed in 1975 by Dr. Edward Southern of Edinburgh University
  • In this technique, fragmented DNA is bound to a substrate (often a nitrocellulose or nylon membrane), denatured, dried and then exposed to a labeled hybridization probe in an appropriate buffer
  • Blot is then extensively washed and analyzed by Xray film, autoradiography or membrane chromogen detection, depending on the type of probe label employed
  • Southern blotting has been largely replaced by newer molecular techniques but it has value in analyzing several trinucleotide repeat syndromes (Fragile X syndrome, Huntington chorea), where the length of the expanded DNA is greater than the usual amplification ability of PCR

  • Array technology was used by Augenlicht et al. in 1984 to analyze retroviral long terminal repeat (LTR element expression in murine colon tumors (J Biol Chem 1984;259:1842)
  • In 1987, Kulesh et al. used arrays to analyze the expression of more than 2,000 different genes constructed from a human fibrosarcoma cell line, with and without interferon treatment (Proc Natl Acad Sci USA 1987;84:8453); the different mRNA derived cDNAs were spotted onto filter paper and analyzed; 29 sequences were induced by interferon treatment
  • Miniaturized microarrays were introduced in 1995 (Science 1995;270:467)
  • First complete eukaryotic genome was placed on microarray in 1997, when Lashkari et al. placed a maximum of 2,470 open reading frames on a glass slide and analyzed total mRNA expression (cDNA) in S. cerevisia, examining the effects of heat and cold shock and culture in glucose vs galactose on global gene expression profiles (Proc Natl Acad Sci USA 1997;94:13057)

  • Since its first research use in the 1980s, the development of better surface technologies, more powerful robots for arraying, better nucleic acid dye labeling techniques and improved computational power and automated analyzers have vastly improved the power and efficiency of microarray, while also lowering the cost of these analyses
  • Microarray is currently used to analyze many different systems, including the classification of microbes and human microbial pathogens, cellular responses to pathogens, drug and toxic exposures, tumor classification, single nucleotide polymorphism detection, the detection of gene fusions, comparative genomic hybridization, alternative splicing detection (exon junction array / exon arrays) and gene expression profiling via analyzing global mRNA levels
  • Most microarray protocols use reverse transcriptase to convert mRNA into cDNA, as DNA is more stable with RNA
Considerations in microarray use
  • Researcher should ask if microarray technology is really necessary for a specific research application because:
    • Microarray is time consuming and slow to setup, difficult to validate, expensive and requires great analytical expertise
    • Microarray can give an enormous amount of irrelevant data
    • Available chip may already have the desired set of gene sequences
    • Multiplex PCR or a custom chip may be a better method of analysis than employing a genome wide array
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