Methods (molecular, IHC, frozen)

Topic Completed: 1 July 2010

Minor changes: 23 March 2021

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PubMed Search: Microarray[TI] molecular[TI] free full text[sb], Microarray[TI] basic procedures free full text[sb], 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. website. Accessed April 15th, 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)
    • 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
Basic procedures

Basic procedures
  • Although there are many different materials and variations commonly used in microarray, the basis of the technique involves the binding of nucleic acid "probes" or "capture molecules" to a solid support
  • These nucleic acids are often chemically synthesized oligonucleotides or cDNAs derived from mRNA, which are labeled with an appropriate fluorescent or luminescent labeled probe, such as Cy3, Cy5 or dye doped silica nanoparticles
  • cDNA probes are usually 25 - 65 base pairs and often derived from the 3' end of mRNAs, as many of these sequences were captured from transcripts using polyT primers
  • Oligonucleotide probes are usually larger, often 1,000 nucleotides

Measuring gene expression
  • Measuring gene expression via cDNA microarray is referred to as "expression analysis" or "expression profiling"
  • Labeled nucleic acid is bound via nucleic acid derivatives attached to the solid support by S-carboxymethyl-L-cystein, polyacrylamide, 3-aminopropyltrimethoxysilane, 3' glycidoxy propyltrimethoxysilane, phenylisothiocyanate, 1-ethyl=3-(3-dimethylaminopropyl)-carbodiimide hydrochloride or other compounds
  • Linkages themselves must be freely soluble in the hybridization and washing buffers, chemically stable and long enough to prevent inhibition of probe target nucleic acid hybridization by steric hindrance
  • Microarray can use up to four different labels; gene expression profiles use one or two probes
  • When one probe in employed, the sample (treated) and control (untreated) are hybridized to separate probe sets and compared; when the sample and control are differently labeled, a single slide is used

  • Solid support is commonly a glass slide, silicon biochip or nylon membrane, often called a gene chip
  • Bound nucleic acid probes may be synthesized oliogonucleotides, cDNAs or more rarely RNA
  • Typically the amount of each bound probe is a few picomoles
  • Each probe hybridizes with a different fluorescently labeled target nucleic acid species, such as an mRNA derived cDNA
  • Hybridization occurs via complementary Watson-Crick hydrogen bond formation between the A:T and G:C pairs
  • Binding specificity is increased with increased bp length of the probe target hybridization sequence, a high probe target G:C content and bp complementarity and if possible, the same / similar probe target Tms (sometimes be difficult with shorter cDNA derived probes, usually not a problem with longer oligonucleotide probes which usually have similar Tms)
  • In some cases mixing is employed
  • Mixing can be done by magnetic bar stirring, air driven bladders or centrifugal / shear mixing
  • Mixing often requires some sample dilution, however it reduces hybridization time, increases signal, lowers background and provides homogeneous hybridization conditions
  • Ratio of labeled target to probe is very high in microarray so high that the amount of target that actually hybridizes with the probe is less than 1% of the total target molecules
  • Thus probe target hybridization does not result in significant target dilution which could alter assay results

Maintaining high sensitivity and specificity
  • Once binding is completed, the chips are extensively washed under stringent conditions, resulting in only exact or near exact Watson-Crick base pairing
  • High specificity is further achieved by having the hybridization step occur at a relatively high temperature, with a low buffer salt concentration
  • High stringency prevents the binding of noncomplementary strands, hairpin formation (probe or target self hybridization) and the disassociation of strands with very high complementary
  • High sensitivity is achieved by the target nucleic acid sequences being highly concentrated
  • Hybridization of a labeled target sequence reveals target binding only
  • Size, sequence and composition of the target are unknown

  • Labeled target sequences are measured via scanning, with images analysis performed by specialized software
  • Fluorescent intensity is measured and quantified, with the darkest areas (pixels) equal to no signal, the brightest or "whitest" areas recorded as maximum intensity and intermediate "gray" areas given corresponding signal intensity
  • Defining the areas to be quantified is difficult and can be done by the user circling the spot or by the software program
  • Once a spot is defined, the total signal intensity is summed and divided by the number of pixels within the spot to give the total signal intensity
  • Average background of the slide is often subtracted from the value of each spot to give initial data
  • Final data is derived from various statistical analytic methods
  • Microarray results show only the relative gene expression levels, not the absolute amount of a gene expression
  • It is also a limited "snapshot" of gene expression patterns and multiple microarray studies or other molecular methods are required to examine changes in gene expression patterns over time
  • Additionally, it is often prudent to verify microarray results with other techniques (PCR, Northern blotting or RNase protection assays are often used)

Common areas
  • Like any other technique, microarray is subject to several common errors

Assay complexity
  • Cloning and PCR steps required to create and process up to one million different sequences, combined with printing these sequences on the microarray chip, is extremely complex
  • Any error in this process will result in the misidentification of an expressed sequence, giving false data

Signal variation and analysis
  • Hybridization step, washing and pixel quantification steps are complicated by many factors, including background fluorescence, uneven hybridization, fluorophore inactivation by ozone and light exposure, temperature variation, cover slip positioning, hybridization time, uneven hybridization and dye leaking giving a false signal
  • Many other small but important details can significantly alter microarray results
  • Locating and troubleshooting these problems can be complex, expensive and time consuming

Incomplete oligonucleotide and cDNA synthesis
  • Extreme care must be taken to insure the quality and sequence integrity of the microarray probes because unrecognized incomplete or altered probes will drastically alter the hybridization step, invalidating assay results
  • Sheer number of probes required in many microarray assays can make this insuring the integrity of the probes very difficult

Data analysis and evaluation
  • Each microarray data set can consist of several million data points
  • Additionally, each microarray result is typically repeated multiple times, giving an enormous amount of raw data to be analyzed
  • Microarray experimental validity rests on multiple complex steps, such as defining the areas to be pixilated and counted, setting and subtracting out the appropriate background level, setting the boundaries of expression significance, compiling the results of multiple experiments and choosing the most appropriate method of statistical analysis
  • Any error at one of these steps will compromise the final data
  • Background intensity analysis can be especially complex in high density microarrays, compared to spotted microarrays, as the former lacks spaces between the probes

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

  • 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

  • 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

  • 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

  • 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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