This is the first installment in the DNA microarray series where I will introduce the technology and explain the basics of the technique to give you a quick immersion if you are new to the subject, and maybe a refresher if you are an old hand at it.
Expressing the story of life from the genetic code
While humans contain about 25,000 genes, only a fraction of these are actively expressed as mRNAs at any one time. This makes sense, since if all the genes were expressed all at once, we would get a garbled stream of unstructured information. Just like forming words in a sentence, expression of the genetic code reflects the specific context and the exact message that needs to be stated.
When we talk, we structure our words into a specific string that forms a sentence. Sentences could be arranged into a paragraph, paragraphs into passages and so on. However, there must be a specific context and purpose to each sentence.
Similarly, specific genes are expressed under corresponding conditions to “update” the functional content of the organism, to respond to internal and external stimuli, resulting in growth, development, and survival. The set of all RNA molecules expressed in a cell at any given time, what it is “saying” at that time, is called the transcriptome.
The development of DNA microarray technology in mid 1990s allowed for the first time to simultaneously profile and study the transcriptome, in other words to study cells’ real-time “chatter” in more detail. The technology exploited the very same principle that makes nucleic acid so essential to information storage: hybridization to complementary sequences.
Hybridization between the cDNA reverse transcribed from a biological sample to a pre-designed complementary DNA probe arranged on a slide, or array, is the basis of DNA microarrays. A microarray therefore consists of a pre-designed library of synthetic nucleic acid probes that are immobilized and spatially arrayed on a solid matrix. Microarrays evolved from a technique known as Southern blotting, where DNA fragments are attached to a substrate and then probed with a known gene sequence.
The first DNA arrays were constructed by immobilizing cDNAs onto filter paper. However, it was not until 1995 that the first DNA microarrays capable of analyzing thousands of sequences were constructed by “spotting”, or attaching short synthetic probes to designated locations on the solid surface, usually glass or silicon chip (see Figure 1).
There are several ways that such spotted arrays can be produced. Some methods basically use a robot to “print” pre-designed probes that have been attached to fine needles onto a chemical matrix surface using surface engineering (examples include fine-pointed pins, needles and ink-jet printing). Other methods employ photo-activated chemistry and masking to synthesize probes one nucleotide at a time on a solid surface in repeated steps to build up probes of specific sequence in designated locations.
Figure 1: A DNA microarray is a collection of synthetic DNA probes attached to designated location, or spot, on a solid surface. The resulting "grid" of probes can hybridize to complementary "target" sequences derived from experimental samples to determine the expression level of specific mRNAs in a sample.
DNA Microarray measurement of gene expression
A basic protocol for a DNA microarray is as follows:
Isolate and purify mRNA from samples of interest. Since we are interested in comparing gene expression, one sample usually serves as control, and another sample would be the experiment (healthy vs. disease, etc)
Reverse transcribe and label the mRNA. In order to detect the transcripts by hybridization, they need to be labeled, and because starting material maybe limited, an amplification step is also used. Labeling usually involves performing a reverse transcription (RT) reaction to produce a complementary DNA strand (cDNA) and incorporating a florescent dye that has been linked to a DNA nucleotide, producing a fluorescent cDNA strand. Disease and healthy samples can be labeled with different dyes and cohybridized onto the same microarray in the following step. Some protocols do not label the cDNA but use a second step of amplification, where the cDNA from RT step serves as a template to produce a labeled cRNA strand.
Hybridize the labeled target to the microarray. This step involves placing labeled cDNAs onto a DNA microarray where it will hybridize to their synthetic complementary DNA probes attached on the microarray. A series of washes are used to remove non-bound sequences.
Scan the microarray and quantitate the signal. The fluorescent tags on bound cDNA are excited by a laser and the fluorescently labeled target sequences that bind to a probe generate a signal. The total strength of the signal depends upon the amount of target sample binding to the probes present on that spot. Thus, the amount of target sequence bound to each probe correlates to the expression level of various genes expressed in the sample. The signals are detected, quantified, and used to create a digital image of the array.
If we are trying to calculate relative expression between two samples, each labeled with a different dye (See figure 2, red for experiment, green for control), the resulting image is analyzed by calculating the ratio of the two dyes. If a gene is over-expressed in the experimental sample, then more of that sample cDNA than control cDNA will hybridize to the spot representing that expressed gene. In turn, the spot will fluoresce red with greater intensity than it will fluoresce green. The red-to-green fluorescence ratio thus indicates which gene is up or downregulated in the appropriate sample.
Microarray technology propelled functional genomics, a discipline that strives to identify the role of genes in cellular processes, into the spotlight because it allowed functional analysis of genome-wide differential RNA expression between different samples, states and cell types to gain insights into molecular mechanisms that regulate cell fate, development, and disease progression. Microarray data is used to generate a profile of gene expression, which serves as a determinant of protein levels and therefore cellular function between biological samples. A single experiment can provide information on the expression on thousands of genes, virtually the entire human genome, to compare expression patterns between any two states. Microarray experiments can indicate which genes are up or down regulated between samples from normal and diseased tissue, or two samples in absence and presence or a certain stimuli. It is easy to see why this technology might be appealing for understanding complex biological systems as well as drug discovery, disease diagnosis, novel gene identification.
In the next installment in this Introduction to DNA Microarrays series, I’ll discuss various types of Oligonucleotide expression arrays, how they are made and their common applications. Stay tuned…
Hope you had a heavy breakfast, because the first qPCR is going to take time. Did you remember to fill your coffee cup? There are a lot of intricacies in qPCR that will need your neural networks to be brisk. How qPCR differs from traditional PCR Unlike traditional PCR, qPCR measures the amplification of DNA […]
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