Methods (molecular, IHC, frozen)
Molecular
Microarray variations


Topic Completed: 1 January 2010

Minor changes: 17 March 2020

Copyright: 2010-2020, PathologyOutlines.com, Inc.

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

Rodney E. Shackelford, D.O., Ph.D.
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Cite this page: Shackelford RE. Microarray variations. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/molecularpathmicroarrayvariations.html. Accessed May 24th, 2020.
Antibody microarrays
  • Different groups of antibodies may be attached to a solid matrix, allowing the microarray to be performed on solutions of proteins, such as cell lysates, urine, plasma or cerebrospinal fluid
  • This assay can be performed with many different variations

Methodology
  • One common method uses the standard enzyme linked immunosorbent assays (ELISA) protocol:
    1. Specific protein I solution is bound by a solid matrix attached antibody
    2. Washing
    3. Binding of a second detection antibody which binds the specific protein
    4. Washing
    5. Addition of a second antibody which has an attached reporter (a fluorophore or horse radish peroxidase [HRP] commonly)
    6. Washing
    7. Specific protein binding is detected via the addition of a HRP substrate or exposure to light wavelengths which cause the fluorophore to emit
Bead based microarray
  • Recently, microscopic polystyrene or latex beads have been employed as the solid support in microarray
  • Beads can carry two or more labels in different ratios creating different bead types, allowing a more complex analysis than microarray performed on a two dimensional matrix
  • Additionally, up to 100 different fluorophores can be used to label 100 different bead attached probes
  • Beads may be placed in a microtitre plate, commonly allowing 96 different samples to be analyzed in parallel
  • Following mixing with labeled target cDNAs, hybridization and washing, the beads can be analyzed by flow cytometry
  • Advantage of this system is that hybridization takes place in solution (instead of a two dimensional surface), often giving a higher specificity for closely related target sequences
Cellular microarray
  • Cellular microarray uses living cells as a target and solid phase probes, which can consist of nucleic acids, antibodies, lipids, proteins, cytokines or any substance to which the cells being examined may bind
  • Like other microarray assays, the target cells are hybridized to the chip, washed extensively and the binding pattern is analyzed
  • Cell count, cell type capture, cell response or changes in cell morphology / phenotype may be analyzed following different treatments applied to the washed chip (staining, morphologic evaluation, etc.)
  • Interesting variation is done with MHC class I or II spotted chips, which allow the analysis of the MHC binding activities of a cell population
Comparative genomic hybridization
  • Comparative genomic hybridization (CGH) is used to detect copy number changes (amplifications or deletions) of relatively large genomic segments
  • CGH only detects unbalanced chromosomal changes; balanced chromosomal alterations, such as inversions and translocations are not detected, as no change in copy number occurs

Methodology
  • Control DNA is taken from cells with a normal karyotype and compared to the sample to be examined
  • Examined sample is often from a tumor or tissue from a child with dysmorphic features who is likely to have unknown genomic amplifications or deletions
  • Originally, CGH was done with fluorescent labeled metaphase chromosomes immobilized on glass slides, cohybridized with different fluorescent labeled control and sample DNA
  • Comparison of the different preparations and fluorescent labels allows the identification of changes in the copy number along specific chromosomal regions
  • Resolution of this approach is low, allowing only changes of 20 Mb or more to be identified (Dufva: DNA Microarrays for Biomedical Research, 2009)
  • Currently array CGH is more commonly used to analyze amplifications or deletions between sample and control cells; it uses an immobilized probe array, to which are hybridized differently labeled normal and sample DNAs; the resolution depends upon the number of immobilized probes employed
  • CGH allows only the comparison of the relative ratio of different DNA segments between samples and controls, not the ploidy; thus, comparing two tetraploid clones lacking amplifications or deletions by CGH would give a normal result
Standard solid phase nucleic acid microarray
  • Nucleic acid based microarrays are based on Watson-Crick nucleic acid hybridization
  • Therefore, any probe may be used to interrogate any sample of labeled nucleic acids
  • Number of different applications of standard chip microarray is enormous and includes exon, siRNA, single nucleotide polymorphism, tumor specific fusion gene and alternative splicing microarrays
Tissue microarray
  • Tissue microarray (TMA) significantly differs from the other techniques discussed in this chapter because the hybridization step involves antibody binding to one target protein or nucleic acid hybridization to one target gene
  • TMA allows for the simultaneous analysis of protein expression in up to 500 different tissue samples

Advantages
  • Relative level of a specific protein's expression can be compared on the same slide, allowing uniform analysis
  • Small amount of tissue is used per sample analyzed, allowing more analyses per same tissue volume and lowering the amount of tissue biopsied from patients
  • TMA is cost effective as performing one TMA is far cheaper than performing many (up to several hundred) separate immunohistochemical analyses on glass slides
  • TMA analysis directly measures proteins levels, thereby avoiding quantifications based on comparing different mRNA species that may be translated with different efficiencies

Methodology
  • To perform TMA, a hollow needle is used to obtain tissue cores as small as 0.6 mm in diameter
  • Usually paraffin embedded tissue from patient biopsies or surgical specimens is used
  • Tissue cores are placed in a specific array within a paraffin block, sectioned, placed on a slide and protein expression is quantified by standard immunohistochemistry
  • Alternatively, FISH analysis can be used to identify the level of specific nucleic acid sequences
  • TMA has been particularly valuable in analyzing protein expression in tumor samples
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