Understanding Microarrays

Ever tried to understand DNA Microarrays and how they work? Well, here is a brilliant multimedia animation to explain the process:

http://gcat.davidson.edu/Pirelli/index.htm

Microarrays are used to measure the gene expression of cells in different conditions. When a cell becomes cancerous, for example, some genes are induced (transcription increases), other genes are repressed (transcription is decreased) and with other genes nothing changes. Cells respond to different conditions, some environmental (e.g. sun burn) and some chemically induced (e.g. taking heroin). Medicines and their affects on gene expression are prime candidates for micro-array analysis so they can find out why people respond differently to the same drug - which genes are up-regulated/down-regulated in the presence of the drug.

Part 1

We'll use the experiment mentioned in the above multimedia example... Yeast cells can grow with or without oxygen. But in order to survive-in or adapt-to these conditions they have to create new proteins and also stop the production of other proteins that are not so useful in that condition.

If we place some cells in one condition (with oxygen) and then extract the mRNA from them we can tell which proteins are being made. Cells in oxygen is our control condition.
If we extract the mRNA from the cells in the other condition (sans oxygen) then we know what proteins are being expressed for this specific state. Cells without oxygen is the experimental condition.

If we compare the proteins being made (or not being made) in the two conditions we can discover what transcription has started or stopped in the anaerobic state.

We use DNA microarrays to do find this out. The microarray chip (glass slide) contains the mRNA of the whole yeast genome attached to it. To make a microarray we need to get the mRNA strands from both samples and use them to make mRNA probes which are attached to the surface of the microarray. This is a long process which we won't go into. Often, a ready-to-use microarray chip is available to buy from a company like Affymetrix.

Part 2

In order to find out which proteins have been transcribed we have to attach the mRNA of the samples in each condition to the microarray and we have to label them in a way to identify which mRNA came from which sample.

Since both the microarray mRNA probes and the mRNA strands from the cells are the same they cannot combine so we have to make complimentary DNA (cDNA) strands from the mRNA of each sample.

The enzyme reverse transcriptase converts the mRNA to cDNA. The cDNA is made with flourescently-labelled nucleotides so under the correct light the cDNA will glow. The cDNA has the complementary sequence of the mRNA, so if the mRNA was as shown below, thecDNA would be:

CUUUUUAUCCCCCGGGC - mRNA
GAAAAATAGGGGGCCCG - cDNA

Sample 1, the control, in aerobic conditions is labelled green. The second sample, the experimental condition, anaerobic, is labelled with a red flourescent. The mRNA is dissolved using RNAse so we end up with pure cDNA.

The red and green cDNA is complementary to the mRNA of the microarray so when it is squirted on to the microarray slide from both samples they quickly bind to their complimentary strands. Anything that didn't attach is washed off.

Part 3

The microarray is scanned using a machine that has two lasers that iduce flourescence from red and green labelled strands. Pictures for each color are stored on the computer and processed to measure the intensity of the flourescence - the greater the intensity, the more cDNA is attached to the probes and this tells us that a particular gene is highly expressed. Or the intensity is really weak so we can tell that that particular gene is barely expressed.The pictures/data can be combined to compare both conditions:

If a gene was expressed only in the control cells then a spot on the microarray would glow green.
If a gene was expressed only in the experimental cells (anaerobic) then a spot on the microarray would glow red.
If the gene was expressed in both conditions the colors green and red would mix to form a yellowy shade.
Genes that aren't expressed in either condition show as black since no light is emitted.

Since each gene of the yeast genome is a spot on the microarray, we know what gene each color-spot represents on the microarray so we can easily find out which genes are induced or repressed after the scan.

Simple sort-of :) . Data retrieved from microarray analysis is usually processed using complex programs like R using the bioconductor module. There's a lot of statistics behind the analysis of the data so most people who deal with this stuff are specialists.

Shaking the foundations of the central dogma (again)

The first shock that came to the creators of the central dogma (Watson & Crick) was the discovery of retroviruses which had a little trick of reverse transcribing their RNA genome into DNA. This was a shock because it was believed that the pathway of DNA to Protein occured in one direction and could not be reversed.

Now a new discovery has shaken the foundations yet again. It has always been believed that three nucleotide bases in DNA, a triplet codon, code for one type of Amino Acid... until now! A marine bacteria Euplotes crassus was discovered where one triplet (UGA) can code for either cysteine or selenocysteine. How the bacterial genome regulates which AA is coded is not clear but it is something amazing.

Read more here: http://scienceblogs.com/notrocketscience/2009/01/one_codon_two_amino_acids_the_genetic_code_has_a_shift_key.php

Edit (30-01-2009): Apprently the two-amino-acids-for-one-triplet-codon thing is not a new phenomena. I just found this in Concepts of Genetics by Klug et al., Ch. 14.6, p361 -

"Only one codon, AUG, codes for methionine, and it is sometimes called the initiator codon. However, when AUG appears internally in mRNA, rather than at an intiating position, unformulated methionine is inserted into the polypeptide chain (instead of the initiator type of methionine, N-formylmethionine (fmet)). Rarely, another codon (in Bacteria), GUG, specifies methionine during initiation, though it is not clear why this happens."

Furthermore, in transcribed mitochondrial DNA (mtDNA), mtRNA, there is another special behavior:

"In human mitochondria, AUA, which normally specifies isoleucine, directs the internal insertion of methionine. In yeast mitochondria, threonine is inserted instead of leucine when CUA is encountered in mRNA..."

In fact, there turned out to be many such examples over time. See table 14.5 on page 362.

It just goes to prove that whatever rule exists there is almost always something that breaks it. It also brings home the complexity and heterogeneity found in biological systems.

Primer Summary

If you bothered to read the primers I wrote before, I salute you. However, I found a better resource that will present the subject much better and it has nice pictures with large text, animations and more! Someone clearly had a lot of time on their hands. It covers Classical Genetics, Molecules of Genetics and Genetic Organization and Control. Here it is: DNA From The Beginning.