Microarray FAQ

The following is meant to help people understand what DNA microarrays are, how they are made, and why they are useful. This is a work in progress, if you have any corrections, clarifications or a new question don't hesitate to email me at sugnet@cse.ucsc.edu.

The Questions:

• What is a microarray?
• how are microarrays made?
• So what are the differences between different types of microarrays?
• how are microarrays used?
• Why ratios and cohybridizations?
• What is a "hybridization"?, how does it work?
• What is "Reverse Transcription"?
• how sensitive are microarrays?
• I still have lots of questions, where can I find more information?

The Answers:

What is a microarray?

A generic microarray consists of multiple features (spots) of DNA which will be used to determine the levels of mRNA expression in a collection of cells. The DNA for each feature is from a gene of interest and is a probe for the mRNA encoded by that gene. In general you can think of a microarray as a grid of DNA spots. Each spot has a unique DNA sequence, different from the DNA sequence of the other spots around it. Thus each spot will hybridize only to its complementary DNA strand. In this way each spot is acting as a probe to determine the levels of a specific mRNA produced by a collection of cells.

The basic idea of using a piece of DNA as a probe to determine the presence of the complementary DNA in a solution goes back a long time. It is the same general technique used in Southern, and Northern blots by molecular biologists every day. The thing that makes microarrays so exciting now is the number of DNA probes that it is possible to place on a microarray. Already there are microarrays with probes for every gene in Yeast, and others with over 19,000 human cDNAs. This allows researchers to observe the response of whole genomes to various stimuli instead of one gene at a time.

To the left is an image of a portion of a microarray that has been hybridized and scanned. Each spot gives information as to the relative abundance of the mRNA which is complementary to the DNA in that spot. In this manner it is possible to gather mRNA expression levels for thousands of genes at the same time in parallel.

how are microarrays made?

Currently microarrays come in many different types but they are only two real fabrication methods.

1. Synthesize DNA probes separately, using PCR for cDNAs or chemical synthesis for oligonuclotides. Then use a robot to spot these DNA probes onto your microarrays into very small grids. The substrate for your microarrays can be glass, plastic, or even nylon membranes. Most labs I know of are using glass microscope slides. This method is compartively cheap and flexible and Pat Brown's lab posted plans to make the robot on the web. Some related technologies use an ink-jet like printer to spray oligonucleotide probes on the microarrays.
   
   
2. Synthesize DNA oligonucleotides directly on the microarray using UV-masks and photo-activated chemistry. Currently Affymetrix are the only people doing this. Affymetrix is a dominant player in commercial microarrays and the chemistry they use is just amazing. The technique used is as follows: deprotect sites that will have the next base (A,C,T, or G) bound to them using UV light, then bind the next base to those sites and repeat with a different base. To direct which sites will be deprotected Affymetrix uses a photolithographic mask which only lets the UV light activate certain sites.
   
   
   
   Using this technology Affymetrix is able to build up very large arrays of oligonucleotides in parallel. however due to synthesis efficiencies the longest oligonucleotide probes that Affymetrix makes are 25 nucleotides long.

So what are the differences between different types of microarrays?

The different types of microarrays each have their own peculiarities and no one that I know of has published any sort of study rigorously comparing the different technologies. however there are some inherent strengths and weaknesses to each technology.

• The Affymetrix chemistry is great, but their technology is very expensive and fairly inflexible. The basic Fluidics station and scanner are over $100,000 and then each GeneChip is around $5,000. The reason that it is inflexible is that if you don't like their arrays and want to make your own the cost for a new photolithographic mask can be over a million dollars. That said, the technology is very robust and Affymetrix's chemistry is reproducible and allows the detection of SNPs and other small features in the DNA.

  One thing to note is that Affymetrix is not currently doing cohybridizations which makes it very important and challenging to normalize between the experimental and control GeneChips. That is to say that Affymetrix does not produce ratios, each probe produces only an absolute intensity.
  
  

• Spotting DNA on glass microscope slides is relatively inexpensive and very flexible. however the spotting process itself is inherently variable. Also most microarrays produced in this manner use cDNAs as their probes. Using cDNAs has a couple of techinical problems.
  1. You need a copy of that DNA to start with so you can use PCR to produce your probes. This is a major point as we know the sequence of whole genomes but we don't have unique cDNA libraries that span genomes.
  2. When using a cDNA as a probe you get a very long sequence to bind to which makes it impossible to discern between genes that are more than 80% similar, and forget about detecting SNPs.
  It is possible to spot oligos on glass slides and save yourself a lot of PCR and avoid the above limitations.

The technique that allows the spotting technology to sidestep the issue of variability in spotting and other concerns is the use of cohybridizations. This technique is covered in greater detail later in this document but the main concept is to use relative RNA expression levels instead of absolute expression levels. To accomplish this two separate RNA samples are used: an "experimental" and a "reference". Each RNA is labeled with a different fluorescent dye, then the two samples are mixed and hybridized at the same time to the microarray. When the microarray is scanned, number of photons in the experimental dye's spectrum is compared to the number of photons in the reference dye's spectrum. Many variations in spot size, probe concentration and other issues are cancelled out in this manner.

how are microarrays used?

The basic protocol is as follows:

1. Isolate the RNA you are interested in and the RNA from your control. The RNA can come from any cells. It is important to realize though that the RNA from tissues or any heterogenous cells may lead to results that reflect changes in the composition of the sample rather than in changes due to the experimental hypothesis.
   
   
2. Label the RNA. Usually this means preforming a reverse transcriptase reaction and incorporating dye that has been linked to a DNA nucleotide. however some protocols, i.e. Affymetrix's, call for an amplification of the RNA and labelling of the RNA itself. For microarrays on nylon membranes usually the label is radioactive.
   
   
3. hybridize the labeled target to the microarray. This consists of placing a solution containing the labeled target on the microarry and letting it sit for a period of hours. This allows a given target to find it's probe on the microarray and bind to it. Usually this is carried out a specific temperature to minimize non-specific binding of target to the probes on the microarray.
   
   
4. Remove the hybridization solution and wash the microarray. The washing can be done at different salt and detergent concentrations to minimize non-specific binding. In general solutions with lower salt concentrations weaken the DNA base paring and are referred to as "more stringent" and vice versa for higher salt concentrations.
   
   
5. Once the microarray has been washed it is time to scan the microarray. Scanning is just quantitizing how much target bound to the DNA probe on the microarray. Most microarrays use fluorescent dyes and are scanned in the following manner:
   
   
   1. laser is used to excite the fluorescent dye, the photons coming from the dye are captured using lenses to focus the light and a photo multiplier tube (PMT) to quanitate how many photons are being captured.
   2. The resulting number for that section of the microaray is translated into one pixel of a 16 bit .tiff file. The more pixels per centimeter, the better the resolution of the resulting .tiff image. It is important to note that .tiff files are uncompressed and file formats like .jpeg and .gif which compress data should not be used for storage of results.
   3. The resulting image is analyzed by finding the spots and comparing the differences between chips (if the hybridization contained only one fluor) or the ratio of the two fluors for cohybridization experiments. how these differences are normalized, compared and interpreted is beyond the scope or this document.
   
   For more information on scanning basics check out Axon's FAQ.

Why ratios and cohybridization?

It can be difficult using microarrays to show that there is a relationship between the absolute intensity of a hybridized probe and the absolute number of target molecules bound. This is especially true with cDNA probes where it may be difficult to control what sequences are present in a probe. Issues such as secondary structure, melting temperature, and even target characteristics make it hard to calibrate an entire array for measuring absolute molecules bound.

In order to sidestep these issues researchers use cohybridizations to measure the level of RNA expression relative to another sample at the same time on the same microarray. The basic idea is as follows:

1. Isolate the RNA from the experimental state and label it with a fluorescent dye, usually Cy5 or Cy3.
   
   
2. Isolate RNA from a control state (also called a reference state) and label it with a different fluorescent dye, usually Cye3 or Cy5, whichever you didn't use for the experimental RNA.
   
   
3. Mix the two samples and hybridize them at the same time on the same microarray.
   
   
4. Wash the microarry to remove non-specific binding and scan the microarray to quantitate the amount of each fluor, both the control and the experimental. The key idea is now to not analyze the absolute intensities, but rather to compare how different the experimental is from the control. This is usually expressed as a ratio of the two numbers.
   
   

There is currently much debate about how to use and normalize these ratios, and even exactly which numbers to use for the ratios. The actual number used to do the ratios comes from the intensities of the pixels that make up the spot of that DNA probe. Once these pixel intensities are adjusted for background there are many ways to extract a single number to use in ratios. Some people use the median pixel value, some use the mean, some researches throw out all of the saturated pixels and then take the mean. I highly reccomend finding a good statistics book and familiarizing yourself with basic statistics agian to understand the significance of these numbers and how they effect your results.

Once you've decided how you want to extract a single value from the pixel intensities you can now measure your ratio and determine if your genes of interest are being expressed more or less relative to your control sample. Some researchers take the log of these ratios as they will then have the nice property of being centered around zero with positive numbers indicating induction a gene and negative numbers indicating repression a gene, relative to the control sample. Keep in mind that there are still a host of issues when it comes to normalizing these values and comparing values between different microarrays. These issues are outside the scope of this document.

What is a "hybridization"?

In order to understand how hybridizations work check out my Biology starter to see the how this technique exploits the marvelous properties of DNA.

What is "Reverse Transcription"?

Reverse transcrition uses and enzyme conveniently named Reverse Transcriptase to produce the complementary DNA strand from an RNA strand. To find out more about the amazing properties of DNA and RNA check out my Biology starter

how sensitive are microarrays?

Affymetrix claims that on a routine basis they can detect 1 molecule in 100,000 and that they can detect two fold changes in RNA expression levels. In an optimal hybridization they claim to detect one molecule in 2,000,000 and to detect 10% changes in RNA expression levels. I haven't seen any published results on spotted arrays but I'd love to hear about it if someone else has.

I still have lots of questions, where can I find more information?

Check out other sources of information or emailme.

The Mguide Build your own and do it yourself.
The Brown Lab FAQ Specific technical questions.
Axon's FAQ. Image scanning quesions.
Check out the Microarray Links page for other resources.