Microarray: Meaning, Production and Types | Biotechnology

In this article we will discuss about Microarray:- 1. Meaning of Microarray 2. What is a Microarray? 3. Why Should I Perform Microarray Experiments? 4. Production 5. Types.

Contents:

1. Meaning of Microarray
2. Why should I Perform Microarray Experiments?
3. Microarray Production
4. Where Can I Obtain Microarrays?
5. Microarray: An Eye Opener
6. Types of Microarrays

1. Meaning of Microarray:

Microarray technologies as a whole provide new tools that transform the way scientific experiments are carried out. The principle advantage of microarray technologies com­pared with traditional methods is one of scale. In place of conducting experiments based on results from one or a few genes, microarrays allow for the simultaneous interrogation of hundreds or thousands of genes.

Microarrays are microscope slides that contain an ordered series of samples (DNA, RNA, pro­tein, tissue). The type of microarray depends upon the material placed onto the slide: DNA, DNA microarray; RNA, RNA microarray; pro­tein, protein microarray; tissue, tissue microarray.

Since the samples are arranged in an ordered fashion, data obtained from the microarray can be traced back to any of the samples. This means that genes on the microarray are addressable. The number of ordered samples on a microarray can number into the hundred of thousands. The typical microarray contains several thousands of ad­dressable genes.

2. Why Should I Perform Microarray Experiments?

The true motivation for performing these ex­periments lies likely somewhere between the two extremes. It is this combination of gene­rating a scientific hypothesis (elegant or not), and at the same time being able to produce massive amounts of data that has made re­search in microarrays so attractive.

For those willing to try this new technology, microarray experiments are performed to answer a wide range of biological questions to which the an­swers are to be found in the realm of hundreds, thousands, or an entire genome of individual genes.

3. Microarray Production:

Printing microarrays in not a trivial task and is both an art and a science. The job requires considerable expertise in chemistry, engineer­ing, programming, large project management, and molecular biology. The aim during print­ing is to produce reproducible spots with consistent morphology.

Early versions of print­ers were custom made with a basic design taken from a prototype version. Some were built from laboratory robots. Current commer­cial microarray printers are available for al­most every size application.

4. Where Can I Obtain Microarrays?

Microarrays can be obtained from a variety of sources. Commercial microarrays are of high quality, good density and available for the most commonly studied organisms including human, mouse, rat, and yeast. Microarray core facilities located in academic and government institutions produce microarrays that are available for use.

If you are fortunate to have your own facility, microarrays can be produced at your own convenience. Microarrays once made store well in dark desiccated plastic slide boxes. Some manufacturers suggest storage at “20°C while others find room temperature ad­equate. The shelf-life of microarrays has been claimed to be up to 6 months although this has not been empirically tested.

5. Microarray: An Eye Opener:

a. Microarrays are based on the principle of hybridization between a “probe” and tar­get molecules in the experimental sample.

b. However, in a microarray the probes are attached to the solid support and the experimental sample is in solution.

c. The microarray represents the genome of the organism being tested and consists of sequences corresponding to each gene in the organism.

d. To monitor gene expression, RNA ex­tracted from a cell sample is tested against a microarray.

e. The experimental RNA sample is usually fluorescently tagged.

f. Hybridization of the mRNA from the ex­perimental sample to the DNA probes on the solid support indicates whether or not a gene is expressed and to what degree.

g. The level of fluorescence at each point on the array correlates with the level of the corresponding mRNA in the sample.

6. Types of Microarrays:

1. DNA Microarray:

Basic Principle:

Complementary sequences of nucleotides stick to, or “hybridize” to, one another. For example, a DNA molecule with the sequence -A-T-T- G-C- will hybridize to another with the se­quence -TA-A-C-G- to form double-stranded DNA.

Types of DNA Microarray:

There are two major types of DNA microarrays. Each type of microarray is manu­factured differently.

(a) cDNA Microarray:

This type contains cDNA fragments 600 to 2400 nucleotides in length. When making a cDNA microarray, each of the different probes must be chosen independently and made by PCR or traditional cloning. Then all the DNA probes are spotted onto the slide.

(b) Oligonucleotide Microarray:

This type uses oligonucleotides of 20 to 50 nucle­otides in length. When making an oligo­nucleotide array, the oligonucleotide is synthesized directly on the slide.

A. cDNA Microarrays:

Steps in cDNA Microarray:

1. Extracting and labelling the RNA Sample:

A typical workflow of the microarray experiment has been summa­rized in Figure below. Once microarrays have been made and obtained, the next stage is to obtain samples for labelling and hybridization.

Labelling RNA for expres­sion analysis generally involves three steps:

(a) Isolation of RNA.

(b) Labelling the RNA by a reverse tran­scription procedure with fluorescent markers.

(c) Purification of the labelled products.

(a) Isolation:

RNA can be extracted from tissue or cell samples by common or­ganic extraction procedures used in most molecular biology labs. Both to­tal RNA and mRNA can be used for labelling, but the contaminating ge­nomic DNA must be removed by DNase treatment.

The amount of to­tal RNA necessary for a single label­ling reaction is about 20 µg while the amount of mRNA necessary is about 0.5 µg. Lesser amounts are known to work, but require extreme purity and well developed protocols.

It is gener­ally a good idea to check the RNA samples before using them in microarray experiments. In fact, for many core facilities it is a requirement. This can be done by assaying the ab­sorption ratio 260/280 lambda and/or running a sample on an ethidium bro­mide stained agarose gel.

(b) Labelling the RNA by Reverse Tran­scription:

Direct Labelling:

Direct labelling of the RNA is achieved by producing cDNA from the RNA by using the enzyme reverse transcriptase and then incorporating the fluorescent labels, most commonly Cy3 and Cy5. Other fluorophores are available (e.g., Cy3.5, TAMRO, Texas red) but have not yet found widespread use.

Indirect Labelling:

In the indirect procedure, a reactive group, usually a primary amine, is incorporated into the cDNA first, and the Cy3 or Cy5 is then coupled to the cDNA in a sepa­rate reaction. The advantage of the indirect method is a higher labelling efficiency due to the incorporation of a smaller molecule during the reverse transcription step.

(c) Purification of the Labelled Prod­ucts:

Once fluorescently labelled probes are made, the free unincorpo­rated nucleotides must be removed. This is typically done by column chro­matography using convenient spin-col­umns or by ethanol precipitation of the sample.

Some protocols’ perform both purification steps. As small a side, ra­dioactivity is still around and may even make a comeback in microarrays. Incorporation of 33P- or 35S-labelled nucleotides into cDNAs have high rates and provide more sensitivity than fluorescently labelled probes.

2. Hybridization:

Conditions for hybridiz­ing fluorescently labelled DNAs onto microarrays are remarkably similar to hybridizations for other molecular biology applications. Generally the hybridization solution contains salt in the form of buff­ered standard sodium citrate (SSC), a de­tergent such as sodium dodecyl sulphate (SDS), and nonspecific DNA such as yeast tRNA, salmon sperm DNA, and/or repeti­tive DNA such as human Cot-1. Other non­specific blocking reagents used in hybridi­zation reactions include bovine serum al­bumin or Denhardt’s reagent. Lastly, the hybridization solution should contain the labelled cDNAs produced from the differ­ent RNA populations.

Hybridization temperatures vary depend­ing upon the buffers used, but generally are performed at approximately 15-20°C below the melting temperature, which is 42-45°C for PCR products in 4X SSC and 42-50°C for long oligos.

Hybridization volumes vary widely from 20 µ1 to several mL. For small hybridiza­tion volumes, hydrophobic cover slips are used. For larger volumes, hybridization chambers can be used. Hybridization chambers are necessary to keep the tem­perature constant and resist the hybrid­ization solution from evaporating.

In small volumes, the hybridization kinet­ics are rapid, so a few hours can yield re­producible results, although overnight hy­bridizations are more common.

3. Scanning:

Following hybridization, microarrays are washed for several min­utes in decreasing salt buffers and finally dried, either by centrifugation of the slide, or a rinse in isopropanol followed by quick drying with nitrogen gas or filtered air. Fluorescently labelled microarrays can then be “read” with commercially available scanners.

Most microarray scanners are basically scanning confocal microscopes with lasers ex­citing at wavelengths specifically for Cy3 and Cy5, the typical dyes being used in experi­ments. The scanner excites the fluorescent dyes present at each spot on the microarray and the dye then emits at a characteristic wavelength that is captured in a photomultiplier tube.

The amount of signal emitted is di­rectly in proportion to the amount of dye at the spot on the microarray and these values are obtained and quantitated on the scanner.

A reconstruction of the signals from each location on the microarray is then produced. For cDNA microarrays one intensity value is generated for the Cy3 and another for the Cy5. Hence, cDNA microarrays produce two-colour data.

Affymetrix chips produce one-colour data, because only one mRNA sample is hy­bridized to every chip. When both dyes are re­constructed together, a composite image is generated. This image produces the typical microarray picture.

Modern cDNA Technology:

Newer technology has been developed to de­crease the variation in size of spotted samples in microarrays. In newer cDNA microarrays, the samples are spotted onto a glass slide using inkjet printer technology. The cDNA samples are sucked into separate chambers of the inkjet printer head, and then spotted onto the glass slide as much as ink is spotted onto paper in a printer.

Inkjet technology prevents variations in size and quantity of cDNA in the sample spots. Special adaptors have been developed to prevent the inkjet sample channels from mixing, thus preventing cross-contamination.

B. Oligonucleotide Microarray:

Oligonucleotides are traditionally synthesized chemically on beads of controlled pore glass (CPG). Therefore, it is not too much of a logi­cal leap to synthesize many different oligo­nucleotides side-by-side on a glass slide.

The main differences between synthesizing single nucleotides on beads versus making arrays on glass slides is that the array has thousands of different oligonucleotides, and each must be synthesized in its proper location with a unique sequence. To accomplish this, photolithogra­phy and solid-phase DNA synthesis are com­bined.

Photolithography is a process used in making integrated circuits, where a mask makes a specific pattern of light on a solid sur­face. The light activates the surface it reaches, while the remaining surface remains inacti­vated.

A glass slide is first covered with a spacer that ends in a reactive group. This is then covered with a photosensitive blocking group that can be removed by light. In each synthetic cycle, those sites where a particular nucleotide will be attached are illuminated to remove the blocking group. Each of the four nucleotides is added in turn.

At each addition, a mask is aligned with the glass slide. Light passes through holes in the mask and activates the ends of those growing oligonucleotide chains that it illuminates. Much as in tradi­tional chemical synthesis, each nucleotide has its 52-OH protected. Thus after each addition, the end of the growing chain is blocked again.

These protective groups are light activated, so at each step, a new mask is aligned with the slide, and light deportments the appropriate nucleotides. The entire process continues for each nucleotide at each position on the glass slide. Making the masks is the key to this tech­nology.

On Chip Synthesis of Oligonucleotides:

Arrays may be created by chemically synthe­sizing oligonucleotides directly on the chip. First, spacers with reactive groups are linked to the glass chip and blocked. Then each of the four nucleotides is added in turn (in this example, G is added first, then T).

A mask cov­ers the areas that should not be activated dur­ing any particular reaction. Light activates all the groups not covered with the mask, and a nucleotide is added to these. The cycle is re­peated with the next nucleotide.

Hybridization on DNA Microarrays:

Hybridization on a microarray is similar to the hybridization of DNA during other hybridiza­tion experiments, such as Southern blots, Northern blots, or dot blots. All these tech­niques rely on the complementary nature of double-stranded DNA.

When two complemen­tary strands of DNA are near each other, the bases match with their complement, that is, thymine with adenine, and guanine with cy- to sine. On a DNA microarray, hybridization is affected by the same parameters as in these other techniques. How the DNA is attached to the slide can affect how well the probe DNA and target DNA hybridize, especially for oli­gonucleotide microarrays.

The short length of oligonucleotides requires that the entire piece be accessible to hybridize. The length of the spacer between the oligonucleotides and the glass slide optimizes hybridization. An oligo­nucleotide attached with a short spacer has many of its initial nucleotides too close to the glass and inaccessible to incoming RNA or DNA.

Oligonucleotides with longer spacers may fold back and tangle up; therefore, again the sequence is inaccessible for hybridization. Oligonucleotides attached with medium-sized spacers are far enough from the glass, but not so far as to get tangled. Thus medium-sized spacers give the best accessibility for hybri­dization.

Hybridization of two lengths of DNA (or RNA with DNA) requires certain sequence fea­tures. One important property is the relative number of A:T base pairs versus G:C base pairs. Because G:C base pairs have three hy­drogen bonds holding them together, it takes more energy to dissolve the bonds. A:T base pairs have only two hydrogen bonds and re­quire less energy.

Thus more GC base pairs give stronger hybridization. If the sequence has many A:T base pairs, the duplex may form slowly and be less stable. Another important consideration is secondary structure. If the target, the duplex may not form efficiently.

All these issues must be addressed when making an oligonucleotide microarray. Computer pro­grams are available that identify suitable re­gions of genes with sequences that will pro­duce effective probes.

Application of DNA Microarrays:

DNA microarrays are used to determine:

1. The expression levels of genes in a sample, commonly termed expression profiling.

2. The sequence of genes in a sample, com­monly termed mini-sequencing for short nucleotide reads, and mutation or SNP analysis for single nucleotide reads.

2. Protein Microarrays:

A different but conceptually similar approach is being applied directly to proteins. Scientists have developed microarrays that can be used either to identify and quantify thousands of different proteins at once or to find associa­tions between different kinds of proteins and between proteins and other molecules. These types of arrays are collectively referred to as protein arrays.

Protein-detecting arrays may be divided into those that use antibodies and those based on using tags. In the ELISA assay, antibodies to specific proteins are attached to a solid sup­port, such as a micro-titre plate or glass slide. The protein sample is then added and if the target protein is present, it binds its comple­mentary antibody. Bound proteins are detected by adding a labelled second antibody.

Another antibody-based protein-detecting array is the antigen capture immunoassay. Much like the ELISA, this method uses anti­bodies to various proteins bound to a solid surface. The experimental protein sample is iso­lated and labelled with a fluorescent dye. If two conditions are being compared, proteins from sample 1 can be labelled with Cy3, which fluoresces green, and proteins from sample 2 can be labelled with Cy5, which fluoresces red.

The samples are added to the antibody array, and complementary proteins bind to their cog­nate antibodies. If both sample 1 and 2 have identical proteins that bind the same antibody, the spot will fluoresce yellow.

If sample 1 has a protein that is missing in samples 2, then the spot will be green. Conversely, if sample 2 has a protein missing from sample 1, the spot will be red. This method is good for comparing protein expression profiles for two different conditions.

In the third method, the direct immunoas­say or reverse-phase array, the proteins of the experimental sample are bound to the solid support. The proteins are then probed with a specific labelled antibody. Both presence and amount of protein can be monitored.

For ex­ample, proteins from different patients with prostate cancer can be isolated and spotted onto glass slides. Each sample can be exam­ined for specific protein markers or the pres­ence of different cancer proteins. The levels of certain proteins may be related to the stages of prostate cancer. This immunoassay helps researchers to decipher these correlations.

Problem with Immune Based Arrays:

The main problem with immuno-based arrays is the antibody. Many antibodies cross-react .with other cellular proteins, which generates false positives. In addition, binding proteins to solid supports may not be truly representa­tive of intracellular conditions. The proteins are not purified or separated; therefore, samples contain very diverse proteins.

Some proteins will bind faster and better than oth­ers. Also, proteins of low abundance may not compete for binding sites. Another problem is that many proteins are found in complexes, so other proteins in the complex may mask the antibody binding site.

Protein Interaction Arrays:

Rather than using antibodies, protein inter­action arrays use a fusion tag to bind the pro­tein to a solid support. The use of protein ar­rays to determine protein interactions and protein function is a natural extension of yeast two-hybrid assays and co-immunoprecipitation.

Protein arrays can assess thousands of proteins at one time, making this a powerful technique for studying the proteome. Protein arrays are often used in yeast because its proteome contains only about 6000 proteins. Libraries have been constructed in which each protein is fused to a His 6 or GST tag.

The proteins are then attached by the tags to a solid support such as a glass slide coated with nickel or glutathione. To build the array, each pro­tein is isolated individually and spotted onto the glass slide. The tagged proteins bind to the slide and other cellular components are washed away. Each spot has only one unique tagged protein. Once the array is assembled, the pro­teins can be assessed for a particular function.

The direct immunoassay binds the protein samples to different regions on a solid support. Each spot has a different protein sample. Next, an antibody labelled with a detection system is added. The antibody binds only to its target protein. In this example, the antibody recog­nizes only a protein in patient samples 1 and 2.

In the laboratory of Michael Snyder at Yale University, the yeast proteome has been screened for proteins that bind calmodulin (a small Ca²⁺ binding protein) or phospholipids. Both calmodulin and phospholipid were tagged with biotin and incubated with a slide coated with each of the yeast proteins bound to the slide via His 6- nickel interactions.

The biotin-labelled calmodulin or phospholipid was then visua­lized by incubating the slide with Cy3-labelled streptavidin. (Streptavidin binds very strongly to biotin). The results identified 39 different calmodulin binding proteins (only six had been identified previously), and 150 different phos­pholipid binding proteins.

Application of Protein Microarrays:

Protein microarrays are measurement devices used in biomedical applications to determine the presence and/or amount (referred to as relative quantitation) of proteins in biological samples, such as blood. They have the potential to be an important tool for proteomics research.