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DNA microarray (gene chip)

Indications: - *expression profiling of malignancies a) prognosis for developing metastasis in axillary node- negative breast cancer [2] b) acute leukemias [6] c) myelodysplastic syndrome [8] d) ovarian carcinoma [9] e) pancreatic carcinoma [9] - assessing fetal chromosomal status* [11] - *prenatal diagnosis in pregnant women with fetal anomalies detected on ultrasound [12] - investigating causes of stillbirth* [11] - post-natal testing to predict developmental delay & intellectual disability in children [13] * uncertain clinical significance [11] Methods: A miniturized chip with thousands of different DNA sequences in tiny wells that permits simultaneous study of multiple gene expression. The tiny wells are typically circular spots with a diameter of 100 um. There are 2 types of array formats: 1) oligonucleotides arrays 15-25 base pairs 2) spotted arrays that contain larger DNA fragments from a) polymerase chain reaction (PCR) b) genomic clones (100 base pairs or greater) c) large oligonucleotides (50-75 base pairs) Arrays may be glass or membrane. Array size may be up to 30,000. Glass slides: 1) standard glass chip or slide size is 1 x 3 inches 2) surface coatings for glass slides to maximize DNA coupling to surface a) aminoalkylsilane* b) poly-L-lysine c) 1,4-phenylene diisothicyanate * silanated glass produces highest signal intensity, lowest background fluorescence, most reproducible DNA attachment In most experiments the gridded DNA is called the probe & the applied hybridization mixture is called the target. [4,5] Standard protocol recommends > 5 ug of total RNA (100-300 ng of polyA+ RNA) as starting material [5]. 25-50 ug of total RNA (1-2# ug of polyA+ RNA) is recommended in ref [4]. Protocols starting with 10 ng of RNA have been described [5]. # a better signal is generally obtained using 2 ug of polyA+ RNA The lower limit of detection in most cDNA microarray systems is 1/100,000 to 1/250,000. This corresponds to single copy messages in single cells, but may not be detected in complex tissues with multiple cell types.

Related

DNA microarray scanner

Specific

Affymetrix CytoScan Dx Assay GeneChip (Affymetrix) mRNA expression analysis by microarray

General

microarray molecular diagnostic test

Figures/Diagrams

Overview of cDNA microarray process

References

  1. Journal Watch 20(21):170-71, 2000
  2. Journal Watch 22(5):40, 2002 van't Veer LJ et al, Nature 415:530, 2002
  3. Microarrays for the Neurosciences, Geschwind DH & Greg JP (eds), MIT Press, Cambridge MA, 2002
  4. Karsten SL & Geschwind DH, unpublished (2002)
  5. Affymetrix Technical Note (http://www.Affymetrix.com
  6. Cole K, Truong V, Barone D, McGall G. Direct labeling of RNA with multiple biotins allows sensitive expression profiling of acute leukemia class predictor genes. Nucleic Acids Res. 2004 Jun 17;32(11):e86. Print 2004 Jun. PMID: 15205470
  7. Grant GM, Fortney A, Gorreta F, Estep M, Del Giacco L, Van Meter A, Christensen A, Appalla L, Naouar C, Jamison C, Al-Timimi A, Donovan J, Cooper J, Garrett C, Chandhoke V. Microarrays in cancer research. Anticancer Res. 2004 Mar-Apr;24(2A):441-8. PMID: 15152942
  8. Pellagatti A, Esoof N, Watkins F, Langford CF, Vetrie D, Campbell LJ, Fidler C, Cavenagh JD, Eagleton H, Gordon P, Woodcock B, Pushkaran B, Kwan M, Wainscoat JS, Boultwood J. Gene expression profiling in the myelodysplastic syndromes using cDNA microarray technology. Br J Haematol. 2004 Jun;125(5):576-83. PMID: 15147372
  9. Muminova ZE, Strong TV, Shaw DR. Characterization of human mesothelin transcripts in ovarian and pancreatic cancer. BMC Cancer. 2004 May 12;4(1):19. PMID: 15140265
  10. Futschik ME, Sullivan M, Reeve A, Kasabov N. Prediction of clinical behaviour and treatment for cancers. Appl Bioinformatics. 2003;2(3 Suppl):S53-8. PMID: 15130817
  11. Wapner RJ et al. Chromosomal microarray versus karyotyping for prenatal diagnosis. N Engl J Med 2012 Dec 6; 367:2175 PMID: 23215555 http://www.nejm.org/doi/full/10.1056/NEJMoa1203382 - Reddy UM et al. Karyotype versus microarray testing for genetic abnormalities after stillbirth. N Engl J Med 2012 Dec 6; 367:2185 PMID: 23215556 http://www.nejm.org/doi/full/10.1056/NEJMoa1201569
  12. American College of Obstetricians and Gynecologists Committee on Genetics. The use of chromosomal microarray analysis in prenatal diagnosis. Committee Opinion No. 581. Obstet Gynecol 2013 Dec PMID: 24264715 http://www.acog.org/Resources_And_Publications/Committee_Opinions/Committee_on_Genetics/The_Use_of_Chromosomal_Microarray_Analysis_in_Prenatal_Diagnosis
  13. FDA News Release: Jan. 17, 2014 FDA allows marketing for first of-its-kind post-natal test to help diagnose developmental delays and intellectual disabilities in children. http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm382179.htm