Microarrays/DNA chips
Microarrays are a relatively new technology that allow scientists to identify which specific genes a cell is using at a particular point in time.
What makes cells different from each other?
Microarray robot
Microarray technology has enabled the automated identification of genes that are being expressed.
Cells use DNA as the code to make proteins. The proteins determine what the cell looks like and what it does.
All of your cells (with a few exceptions) have the exact same copy of DNA. Yet your skin cells look and act differently from your muscle cells, which are different from your bone cells, which are different from your blood cells? What makes all of these cells look and function differently?
The pieces of DNA that are used to code for proteins are called genes. Only some of the genes are used ('expressed') in each cell. Scientists are interested in finding out which genes are being expressed by different kinds of cells under specific conditions, and they can now use microarrays to do this.
The idea behind microarrays
Microarrays: What are they and why are they useful?
Cells contain thousands of genes but not every gene is used, and different combinations of genes are used at different times. Microarrays allow scientists to work out which genes are being used by different cells. Why is this so useful?
Microarrays: What are they and why are they useful?
Cells contain thousands of genes but not every gene is used, and different combinations of genes are used at different times. Microarrays allow scientists to work out which genes are being used by different cells. Why is this so useful?
When cells are actively using (or 'expressing') particular genes to make particular proteins, messenger RNA (mRNA) is made as an intermediate step. By collecting and analysing all of the mRNA in a cell, you are able to tell which genes are being expressed by that cell at that specific instant in time. Scientists use microarrays to do this.
Identifying genes being used in cells is not new science. It was done before microarrays were available, but only one gene (or a small group of genes) could be studied at a time. Microarrays allow large-scale screens for gene activity.
Collecting the mRNA sample
Scientists collect the mRNA from a cell by breaking open ('lysing') the cells and centrifuging the solution to achieve a crude separation. The "layer" containing the DNA and RNA is then passed through a chromatography column.
The beads of the column have lots of thymine (T) molecules on them. mRNA molecules all have a repeat sequence of adenine (A) nucleotides on the end (a 'poly-A tail'). The A's of the mRNA stick to the T's on the column, but everything else passes through. The mRNA molecules can then be eluted (washed out) of the column. In this way, mRNA is isolated (separated) from the other cellular components.
Making a DNA copy
Setting up a microarray experiment
A number of steps are involved, including isolating the mRNA (produced by transcription), making copies (cDNA) and then analysing these.
Because mRNA is unstable and breaks down easily, scientists must make DNA copies of the mRNA that has been collected. The DNA that is made is called complementary DNA (cDNA). It is made by a process called reverse transcription. During this process, each cDNA molecule is also "labelled" with a fluorescent dye.
Comparing two different cells
Scientists are often interested in comparing the gene expression of different cells to see which genes are "switched on" in each cell - how they are the same and how they differ. For example, scientists might want to find out which genes are expressed in a normal cell versus a cancer cell (to identify cancer-specific gene activity), or in fruit cells in different stages of ripening (to identify gene activity associated with fruit development).
To carry out a comparative experiment, mRNA is collected from both types of cells (and kept separate in two different solutions). cDNA copies are then made. The cDNA reresenting the two different sources is labelled with fluorescent dyes of different colours (to be able to differentiate between the two sources).
Working out which genes are expressed
At this stage, the scientists still only have a collection cDNA samples - perhaps thousands of different ones from each cell type. Their next task is to identify which specific genes have been expressed. This is where a microarray is used. A microarray contains tiny fragments of known DNA sequences in different spots on a slide.
Microarrays: The high-tech machinery
Robots and biotechnology research: Because the microarray process uses DNA and RNA molecules, which are tiny, sophisticated robots are required. Dr Bart Janssen from HortResearch shows off some of the machines and explains what they do.
Microarrays: The high-tech machinery
Robots and biotechnology research: Because the microarray process uses DNA and RNA molecules, which are tiny, sophisticated robots are required. Dr Bart Janssen from HortResearch shows off some of the machines and explains what they do.
Next, liquid solutions of cDNA (labelled with fluorescent dye) from the two or more cell types under investigation are put onto the slide. The cDNA pieces will bond by complementary base pairing to the corresponding gene sequence.
Finally, the slide is washed so that the unbonded cDNA pieces are removed.
Microarrays are made by taking the entire DNA sequence (the 'genome'), breaking it apart, and attaching the fragments to the glass slide in a way so that they are strongly bonded and won't wash off. A specialised robot isused to do this (see the figure). In some cases, pre-prepared microarrays for specific genomes can be bought from scientific suppliers.
Now the slide can be viewed under a powerful, computerised microscope. You can see where the cDNA has bonded to the microarray by the colour that shows up. In the example below, cDNA of one cell type (e.g. a normal cell) shows up as red. cDNA of the other cell type (e.g. a cancerous cell) show up as green. Spots that look yellow have cDNA from both cell types, meaning that both kinds of cells use the gene in that spot. Spots that don't have any cDNA bonded to them are black.
A computer is used to collect and interpret all the data. This is called bioinformatics.
Why are microarray results useful?
Microarray results
Microarray technology has changed the way scientists can investigate and compare gene expression in different cells.
Scientists use microarrays to find out which genes are used by different types of cells. For example, scientists can use microarrays to see:
which genes in a cancer cell are active, compared with normal cells
how cells of the same type respond to changes in the environment (for example, how a diseased cell responds to an added hormone or drug)
which genes are active in the different stages of mitosis (cell reproduction)
which genes are active in development (e.g. fruit development). This can be used to identify key genes/gene pathways, which could then be used as the basis for a screen for future breeding decisions.
