Modules: 2.1 | 2.2 | 2.3 | 2.4 | 2.5 | 2.6
Today you will use one tool, a DNA microarray, to simultaneously examine the expression of many genes. DNA microarrays are slides with DNA sequences spotted in a known order on the surface. The spots of DNA, each one smaller than the period at the end of this sentence, are placed on the slide surface with robotic arms or built one base at a time with photolithography. Each spot of DNA gets a unique address on the slide surface, and the identity and location of each spot get stored in the computerized "design file" for the array. The slide shown below is the same size as the one you'll use (1 x 3 inches) but yours will have 4 arrays each with 44,000 spots of DNA instead of the 250 dots shown!
DNA microarrays are slides with DNA sequences.
Consider the two spots highlighted on the microarray shown above (an array of human genes for this example). The first spot, in Row 6 Position 30, is a 60-nucleotide sequence from the human gene for glyceraldehyde-3-phosphate dehydrogenase (GAPD). This gene, which encodes an essential metabolic enzyme, has been called a "housekeeping" gene since it must be expressed in all human cells no matter how specialized. Other housekeeping genes include those for ACTB (encoding a cytoskeletal protein), TBP (encoding a general transcription factor), HPRT (encoding an enzyme required for nucleotide transport and metabolism), and PPIA (encoding an enzyme important for protein folding). The second spot highlighted on the array, in Row 4 Position 10, is a 60-nucleotide sequence from the human TERT gene. This gene encodes the protein subunit of telomerase, an enzyme that adds telomere repeats (TTGGGGTTG) to the end of chromosomes. As healthy cells age and divide, telomere repeats are lost. Cancerous cells express telomerase and so the telomeres do not shorten. Consequently, these cells "lose track" of how old they are and become immortal.
The GAPD and TERT spots can be used to illustrate how microarray data is generated and interpreted. Consider a group of "normal" human cells and a cancerous version of them. RNA from each type of cell can be isolated (you've seen how quick and easy it is to isolate RNA), converted from RNA into a complementary strand of DNA (called cDNA), and then "color coded." The most commonly used molecules for color-coding are the green-fluorescing cyanine 3 (Cy3) and the red-fluorescing cyanine 5 (Cy5).
For this example, the normal cells get green and the cancerous cells get red. The two colored samples are mixed and then simultaneously hybridized to a DNA microarray. The DNA spotted on the surface of the slide is in vast excess to either colored cDNA sample and so the intensity of each color will vary with the amount of RNA originally present in each sample. A gene expressed similarly in normal and cancerous cells, like the housekeeping GAPD gene, will give rise to a yellow spot in Row 6 Position 30 since equal amounts of green and red cDNA will be bound there and the merged color will appear yellow. By contrast, only red cDNA will bind at Row 4 Position 10 since cancerous cells express telomerase and normal cells do not.
The GAPD and TERT spots can be used to illustrate how microarray data is generated and interpreted.
NOTE: The cDNAs are not really piled on top of one another on the array. Rather they are hybridized side by side to the spot of DNA that is on the surface of the slide.
With an expensive machine, the slide is "scanned" to measure the intensity of the red and green light at each spot (remember we're talking about thousands of spots!) and the data can then be assessed and normalized. Corrections are often made to account for differences between Cy3 and Cy5 incorporation into the cDNA as well as how much of each fluorescent molecule sticks non-specifically to different areas of the slide. These are things you will do next time with your own data.
Today you will convert the RNA isolated from your mouse cells into cDNA and hybridize the cDNA to a DNA microarray.
Creating cDNA from RNA is done using an enzyme called reverse transcriptase. Like all DNA polymerases, this enzyme can only add sequence to an existing chain and so needs a short "primer" to begin synthesis. To perform the cDNA synthesis, you will use a kit from a company named Genisphere. The primers in this kit have a special "capture sequence" at their 5' end. The capture sequences allow the cDNA to be reacted with Cy3 or Cy5 later.
Creating cDNA from RNA is done using an enzyme called reverse transcriptase.
Before you begin today's protocol, prepare your bench for working with RNA. This involves cleaning your pipetmen, retrieving your "RNA only" pipet tips and solutions and wiping down your bench. You should work on a fresh piece of benchpaper and remember to wear gloves when working with RNA.
Calculate the volume needed for 4 ug of each RNA sample, then assemble the annealing reactions in RNase-free eppendorf tubes according to the following table. If you need more than 10 µl for 4 ug of RNA then you should calculate the mass in 10 µl of the more dilute sample and match that mass for the more concentrated sample. You cannot put a volume of RNA greater than 10 µl in the tubes.
| TUBE A | TUBE B | |
|---|---|---|
| RNA | 4 µg of RNA from the control sample | 4 µg of RNA from the experimental sample |
| RNase-free H20 | Bring volume to 10 µl | Bring volume to 10 µl |
| RT primer | 1 µl Capture Sequence I vial 11, red | 1 µl Capture Sequence II vial 11, blue |
Data for these exercises (XLS)
You can decide to take notes on this in your notebook or to skip it...up to you.
Begin with the sheet called "Sample Data" which contains all the data for 21 of 22,000 spots on a tested yeast array. This sheet will familiarize you with the headings and entries you can expect to see when you examine your own data. Some important landmarks are
Please answer the following questions about this data:
To look at the signal intensities for these 21 spots, you should scroll right. Column AF reports the signal from the green fluorescent molecule Cy3 and Column AG reports the signal from the red fluorescent molecule Cy5. Use the "Format" menu to convert the values in these columns from scientific notation to numbers with no decimal places, and then answer the following questions.
Now you are ready to look at a bigger data set and practice some analytical methods. Look at the second sheet called "Test Array" in the Excel file. This sheet has a subset of the data (9 of the 86 columns) for a subset of the spots (1,500 of the 11,000) from a single microarray experiment.
Some of the data analysis you will perform is
You will begin by "normalizing" the data. Many normalization methods have been suggested since microarray technology was introduced. We will practice a "global normalization" method that assumes the Cy3 and Cy5 fluorescent intensities differ by a constant factor,
R = kG where R = red (Cy5) and G = green (Cy3)
One way to determine k is to label the same RNA sample with either Cy3 or Cy5 and then compare the mean signal intensities observed on an array. Since microarray experiments are expensive to perform, this direct comparison is not often done. Instead it is assumed that arrays have the same amount of total mRNA for two samples and the difference in overall intensity is k.
Because microarrays are physically small, signal artifacts routinely arise. These artifacts come from tiny droplets with fluorescent molecules that remain on the array, and from scratches on the surface of the slide. Even the light that leaks into some scanners can make parts of the array appear more green or more red. The column headings in your spreadsheet that include "BG" have background measurements and these values can be used to correct the signal intensities for background artifacts.
So far you've seen that microarray data must be normalized to correct for Cy3 and Cy5 differences as well as "background subtracted" to correct for artifacts on the slide. Recall that microarray experiments are designed to simultaneously compare the expression of many genes in two samples. The corrected intensities can be expressed as a ratio between the corrected signals for the two samples (Green/Red). A ratio of 4 means 4-fold gene induction and a ratio of 0.25 means four-fold repression of that gene.
To avoid the decimals associated with gene repression, the log2 of the ratios is useful. Four-fold induction is reported at log2(4) = the power of 2 needed to get 4 = 2. Four-fold repression is reported as log2(0.25) = the power of 2 needed to get 1/4 = log2(1) – log2(4) = -2. Log2 transformed data makes more sense graphically since a 4-fold induction and a 4-fold repression have the same value but different signs (i.e. +2 and –2).
The arrays we will use are the mouse whole genome microarrays from Agilent. Each slide has four arrays, each with 44,000 60-mer oligonucleotides. These "oligos" were first built on glass wafers and then printed onto the slide surface. The oligos represent more than 41,000 mouse genes, many spotted on the slide more than once.
The success of your experiment is absolutely dependent on the following:
To hybridize the arrays
Figure: Layout of Agilent 4x44K whole mouse genome array.
| TEAM COLOR | AGILENT SLIDE |
|---|---|
| Red | Slide 1, array 1 |
| Purple | Slide 1, array 2 |
| Blue | Slide 1, array 3 |
| Pink | Slide 1, array 4 |
| Green | Slide 2, array 1 |
| Yellow | Slide 2, array 2 |
When every group is ready, we will walk the arrays over to the BioMicroCenter in Building 68 to put them in the 60°C hybridization oven.
Here's what will happen tomorrow:
During this time, the Cy3 and Cy5 agents will bind the capture sequences on the cDNAs bound to the arrays.
DONE!
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