Microarrays.

I’m writing this post because microarrays are perhaps the coolest bit of molecular biology kit in my opinion. The name itself is self explanatory – an array is a regular arrangement and micro means ‘small’.

Microarrays are arrangements of various things, ranging from probes to tissues on very small surfaces, such as microscope slides for instance.

Affymetrix GeneChip.

Based on what is arrayed on the surface, microarrays can be grouped into discrete groups.

[1] DNA Microarrays.

Here, DNA probes are bound to the surface using covalent modification, these probes specifically bind to complementary single stranded DNA/RNA samples from specific genes. This means that one can check if a particular gene is being expressed or not by seeing if DNA/RNA from a sample binds to a specific probe or not.

The detection of specific DNA sequences using microarrays is relatively simple compared to applications such as expression profiling. The key steps are as follows.

1) Isolate DNA sample from tissue.
2) Denature DNA into single strands (heating is one method)
3) Tag DNA sample with a fluorescent probe.
4) Add to the array and allow complementary sequences to bind.
5) Wash off unbound DNA (non-complementary) and visualize.

Visualised Microarray. I used this array as part of a project looking at differential methylation between head and neck cancer cells and peripheral blood mononucleocytes.

DNA that is complementary to the probes on the array will stay bound and hence show up, allowing detection. Since it is possible to put several thousands of probes for thousands of genes on an array it is easy to scan for their presence at once.

Of course, with slight modifications, it is possible to use microarrays to see which genes are being expressed in a cell. Here, RNA is isolated from the tissue and treated with Reverse Transcriptase, which produces cDNA (complementary DNA) that corresponds to the genes being expressed, cDNA is then put through the same workflow as mentioned above and one can see which genes are being expressed in the tissue sample.

Things can be taken one step further to compare variations in gene expression between different cell or tissue types or normal and diseased cells. In this case one can find out if the expression of a gene is upregulated or downregulated with respect to the reference we are using (which is called a control in scientific parlance)

Differential Expression Analysis Workflow, courtesy Memorial University, Canada.

This is done by isolating cDNA from both the control and the test sample, tagging them each with a different coloured probe, then hybridizing them on the same chip and comparing the intensities of fluorescence for each of these probes, with the composite result yielding a picture of which genes are expressed more in test sample than control and vice-versa.

This kind of procedure is also known as Array Comparative Genomic Hybridization, it can be used to assay changes in copy number too (how many copies of a gene, which translates into amounts of mRNA) to see if a gene is overamplified or deleted in a given sample.

There is an absolutely gorgeous poster on Array CGH and how it is empowering cancer research available in .PDF format from AAAS/Science here

[2] ChIP Arrays.

These are modified versions of DNA microarrays that are used to study the epigenetic status of genes, now genes can be expressed or not based on the status of the methylation of promoters and the presence of associated proteins called histones. It is possible to obtain DNA samples from tissues, isolate those that are associated with histones using an antibody that binds specifically to histones, and to then analyse these DNA samples using microarrays to find out which particular genes are subject to chromatin modification. ChIP, by the way, stands for Chromatin Immunoprecipitation.

This technique can also be used to examine the binding of other proteins such as transcription factors to DNA.

ChIP on Chip Wet lab workflow, Courtesy Wikimedia Commons.

Please click on the image for a larger version.

[3] Tissue Microarrays.

Now these kinds of microarrays are fundamentally different from the types mentioned above. These are meant solely for facilitating microscopic analysis of tissues in a manner that is more comprehensive than traditional histological methods that involve mounting one section per slide.

Tissue Microarray Slide , courtesy Wikimedia Commons.

These arrays consist of up to a thousand tissue cores in a paraffin block mounted on a slide, the idea is that examinations of multiple tissue samples can be carried out in one sweep, if you will. This is called multiplex analysis.

The process for making these is as follows.

1) Isolate tissue cores from biopsy sample using a hollow needle.

2) Embed cores in a paraffin block.

3) Section block into thin sections using a microtome.

4) Mount sections on slide.

This approach is extremely useful because tissue processing can accommodate a wide range of studies, ranging from morphological analysis to immunohistochemical studies (where the presence of certain proteins can be detected with the help of antibodies linked with stain) to fluorescence studies, where proteins can be fluorescently tagged and visualized using a fluorescent microscope.

Fluorescent Microscopy of Tissue Section, courtesy Immunoportal.

Immunohistochemically Stained Section of Bone Marrow, courtesy McGill University, Canada

Cantilever Arrays – A footnote.

Recently there have been advances in array technology that allows for the development of extremely accurate arrays which do not need the sample to be amplified or the sample to be tagged with fluorescent probes et cetera. These arrays are known as Cantilever Arrays because the probe is bound to the tip of a cantilever that can bend when complementary DNA/RNA is present in the sample. This bending results in the reflection of an incident laser beam which can be used for quantification. Now that is simple and desirable at the same time because it greatly simplifies workflows (thus saving costs) while increasing speed and reliability, which should translate to better usability in the clinic.

Cantilever Arrays and Potential Applications, courtesy Swiss Nanoscience Institute, University of Basel, Switzerland

Here is a landmark paper on the subject, titled “Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array” which explains what I typed above in detail. Happy Reading.

Further links and things to do

[1] You can carry out a virtual lab experiment involving differential expression analysis between cancer cells and normal cells at the University of Utah’s Genetic Science Learning Center here. Please do also peruse the additional resources they have linked to.

[2] Watch a video summarizing the basics of Microarray technology.

[3] Read a Scitable article on Genetic Diagnosis and DNA Microarrays in Cancer here

[4] Read a Scitable article on Array CGH here

I hope you have fun reading and learning.

-Ankur.

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2 responses to “Microarrays.

  1. Nakita Forcello

    I know this if off topic but I’m looking into starting my own blog and was intriguing what all is required to get setup? I’m assuming having a blog like yours would cost a pretty penny? I’m not very web savvy so I’m not 100% certain. Any suggestions or advice would be greatly appreciated. Many thanks

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