Comprehensive Notes on Microarrays, Probes, Analysis, and Applications

Microarrays, Probes, Analysis, and Applications

Lecture Overview

This lecture covers the following topics:

The home brew explosion mystery.
Understanding the role and location of probes in microarrays.
Verifying the reliability of microarray data.
Methods for analyzing microarray data.
The scientific explanation behind the beer explosion.
Insights from studying stressed-out yeast.
The reasons behind gene duplication.

Basic Research with DNA Microarrays

What Happened to My Home Brew?

A story about a first-year university student making home brew.
The bottles exploded due to fermentation issues.
Microarrays can provide answers to biological questions like this.

DNA Microarrays

A technique used by biomedical scientists to detect and measure the expression of thousands of genes simultaneously.
Involves a glass slide or chip with numerous single-stranded DNA or RNA fragments (probes) fixed on it.
Developed in the 1990s.
The first microarray was the size of a microscope slide.
Miniature versions are known as DNA chips.
DNA on these chips is stable and doesn't degrade.
Used to simultaneously measure the rates of transcription of every gene in a cell.
DNA chips can contain several 100,000 probes.
Microarrays typically have 15,000-80,000 probes.
Each spot on a microarray contains approximately $10 ng$ of DNA.
PCR amplified fragments are a superior source of probes.
PCR products are suspended in a special 'spotting solution'.

Microarray Printing Technology

Split-pin/tweezer:
- Transfers nanolitre amounts of DNA to the array via capillary action.
Pin-and-loop:
- Picks up DNA in a small loop and stamps it onto a slide at a uniform density.
Ink jet:
- Sprays picolitre droplets under pressure.
Robotic machines (Microarrayers):
- Print single-stranded DNA fragments onto the slide in a tightly spaced grid.

Significance of Microarrays

Microarrays enable one scientist to gather more gene expression information in two days than an army of scientists could collect in several years using older techniques.

Introduction to Microarrays

Where's the Probe?

The basic procedure involves depositing tiny amounts of DNA (probes) onto an array surface of a few square centimetres.
The probes are interrogated by hybridization to a target mRNA that is fluorescently labelled.
Probes are the array of gene sequences to be studied.
DNA probes are fixed onto chips and are interrogated with fluorescently labelled target mRNA.

Steps in Microarray Analysis

Create a DNA chip with thousands of short, single-stranded DNA molecules (probes).
Isolate mRNA from cells or tissue samples.
Use reverse transcriptase and fluorescently labelled nucleotides to create cDNA from the mRNA.
- Review: DNA Replication -> RNA Transcription -> Protein Translation.
- Reverse transcription: mRNA to double-stranded DNA catalyzed by reverse transcriptase.
Hybridization:
- Apply the cDNA mixture to the DNA chip.
- Rinse off excess cDNA and scan for fluorescence.
- Each fluorescent spot indicates that the cDNA strand was complementary to a strand on the DNA chip.
- Identify expressed genes.
- The amount of target that sticks to each spot of probe is proportional to the amount of the transcript in the sample.
- It is detected by the intensity of the fluorescent signal.

How Does It Work? - The Samples

Two samples are used: a control and an experimental sample.
Each sample's mRNA is converted to cDNA and labelled with a different fluorescent dye.
The use of two colors allows for comparison of gene expression between the two samples.
In parallel, a population of cells has its mRNA converted to green cDNA, while another population has its mRNA converted to red cDNA.

Process

RNA extraction from cells.
Fluorescent dye labeling.
Hybridization of labelled cDNA to the DNA microarray.
Quantity of expressed genes is measured based on color strength (luminescence intensity).
- The red and green cDNAs are placed on three chips:
  - RED
  - GREEN
  - RED & GREEN
  - Each chip covered by a glass cover slip and incubated.

Transcriptome

Transcriptome = All mRNA, all the genes expressed.
Results from a single DNA chip: Grey = not expressed. The Red and Green transcriptome shows genes that are expressed in both transcriptomes.

Data Analysis

The standard approach is to compute the ratio of fluorescence intensities of two samples hybridized to the same microarray.
One sample (control) is labeled with a green dye (Cy3), and the experimental sample is labeled with a red dye (Cy5).
The green dye (Cy3) has a different fluorescent spectrum to the red dye (Cy5).
The microarray is exposed to both reference cDNA and experimental cDNA.
Then, it's exposed to lasers in the green fluorescence spectrum and the red fluorescence spectrum.

Microarrays Overview

DNA microarray making
Hybridisation
Results delivery
- Microscope glass slides.
- Strain 1 & 2
- RNA Extraction mRNA amplified by PCR
- Spotting (deposit)
- Cy3 reverse transcription Cy5
- LASER Scanning
- Results analysis

Microarray Data Validation

Verifying Microarray Results

Traditionally, gene expression has been tested using Northern blots.
The intensity of a signal on a Northern blot can be compared to the intensity of a signal on a microarray as a check of microarray technology.
Northern blotting involves:
- RNA extraction.
- Electrophoresis to separate RNA by size.
- Transfer of RNA to a membrane.
- Fixing RNA to the membrane with UV or heat.
- Hybridization with labeled probes.
- Visualization of labeled RNA on X-ray film.

Northern Blot Results

Comparison of Northern blot and microarray data in yeast under different conditions (hours).
Black = equal, red = expressed, green = unexpressed.
Representation of fold repression and fold induction.

Data Analysis

When a DNA array is exposed to cDNA labelled with Cy3 and Cy5, they competitively hybridize.
A range of colours develop, indicating relative levels of expression of each gene.
All green indicates expression in reference tissue and all red indicates expression in the experimental tissue.
In-between colours indicate relative levels of expression.

Application

Develop a gene expression profile as a particular type of cancer develops.
Reference sample is healthy tissue.
Experimental tissue could be a pancreatic tumor from discovery through metastasis and then death.

Process post cDNA chip Acquisition

Expose to two target cDNAs which will hybridize (or not) with the probes in the array.
Target lasers detect the fluorescence of the two fluorescent dyes.
The images are combined to generate an expression profile.

Microarray Technology

Summary table of data for one yeast DNA microarray, including fluorescence intensity for each color and the ratio of red signal to green signal.

The location for each spot is important.

Block	Column	Row	Gene Name	Red	Green	Red/Green Ratio
1	1	1	Tub1	2,345	2,467	0.95
1	1	2	Tub2	3,589	2,158	1.66
1	1	3	Sec1	4,109	1,469	2.80
1	1	4	Sec2	1,500	3,589	0.42
1	1	5	Sec3	1,246	1,258	0.99
1	1	6	Act1	1,937	2,104	0.92
1	1	7	Act2	2,561	1,562	1.64
1	1	8	Fust	2,962	3,012	0.98
1	1	9	Idp2	3,585	1,209	2.97
1	1	10	Idp1	2,796	1,005	2.78
1	1	11	Idh1	2,170	4,245	0.51
1	1	12	Idh2	1,896	2,996	0.63
1	1	13	Erd1	1,023	3,354	0.31
1	1	14	Erd2	1,698	2,896	0.59

Measuring Fluorescence

Some merged images look more red than green, more green than red, or about equal red and green.
Separate red and green channel images.

Gene Transcription Analysis

Understanding how computers read microarrays.
Red-green colour scale for changes in transcription.
Genes that are equally transcribed in both conditions are coded black.
Experimentally induced (increased) genes are colored red.
Experimentally repressed (reduced) genes are colored green.

cDNA Microarray Technology

Conceptually simple method for monitoring levels of gene expression.
PCR-amplified cDNA fragments of genes are spotted at high density on a glass slide/DNA chip at 50-100 spots per $mm^2$ .
This is then probed against fluorescently labelled target cDNAs.
Overview of DNA Microarrays
- Control Cell & Experimental Cell
- mRNA extracted from cell
- Reverse transcription, fluorescently labeled with Cy3 (Green) and Cy5 (Red)
- Combine equal amount and hybridize onto microarray
- Scan

The Beer Explosion Mystery

Why Did the Beer Blow?

Yeast ferment anaerobically, extracting energy from sugar.
$CO_2$ is produced as a waste product, and alcohol is produced as an energy storage compound (though slightly toxic).
Fermentation stops when sugar runs out.
Possible causes of explosion:
- Too much sugar to start with (a lot of $CO_2$ ).
- Presence of oxygen in the beer bottles (too much pressure from oxygen and $CO_2$ ).
Microarray analysis shows that genes responsible for the breakdown of ALCOHOL, to less-toxic storage compounds glycogen and trehalose, are switched ON in the presence of OXYGEN.
More $CO_2$ is produced, thus further metabolism of sugar, leading to the BOOM!
If oxygen caused the explosions, the beer would not have had much alcohol in it, because the alcohol was broken down.

Normal Fermentation (to make alcohol)

Sugar + yeast -> (alcohol & $CO_2$ )
$CO_2$ = waste product (TOXIC)
Alcohol is broken into Trehalose & Glycogen in the presence of oxygen.
If too much $O_2$ to start with, alcohol becomes break down products causing too much pressure.
So, no alcohol produced at all !! Energy Ferment Alcohol & Oxygen

Why Yeast Make Alcohol

Yeast make alcohol, which is toxic, as a temporary energy storage compound.
Yeast that could tolerate alcohol had a competitive advantage over other yeast until oxygen became available.

Stressed-Out Yeast and Gene Copies

What Can We Learn From Stressed-Out Yeast?

Yeast contain similar cell-signaling pathways to humans, without the complexity of cell specialization and tissue formation.
Yeast is a model for cancer research because mitosis is almost identical in all species.
Our understanding of the human cell cycle is due to yeast biologists, because yeast and human cells have many genes in common.
Drosophila is also a good model organism for human inherited diseases.

Why Are There So Many Copies of Some Genes?

Yeast contains genes that encode isozymes, which have a lot of sequence identity and seem redundant in vitro.
However, microarray analysis reveals that these isozymes are often expressed differently, suggesting they have different jobs to do.

Isozyme Location & Function

The location of an isozyme within a cell means that it does do a different job, peculiar to the sub-cellular environment in which it is found.
They may seem to work identically in vitro but act differently in vivo.
There may be multiple environmental triggers for a particular enzyme.
Selection has favored four isozymes with a simpler promoter, rather than one gene with a complex four-transcription-factor promoter.

Isoenzymes

LDH 1 HHHH: Occurs in myocardium (aerobic tissues)
LDH 2 HHHM: In acute leukemia
LDH 3 HHHM: In acute leukemia
LDH 4 HMMM: Occurs in muscle and liver (anaerobic tissues)
LDH 5 MMMM: Occurs in muscle and liver (anaerobic tissues) in liver disease

Medical Relevance

Many diseases are caused by the absence, malfunction, or inappropriate expression of a particular enzyme (e.g., SOD).
Enzymes serve as targets for a variety of drugs.
Enzymes are sometimes administered in the treatment of disease (e.g., Multiple Sclerosis).
The presence or absence of specific enzymes can be used to diagnose specific diseases.
Relationship of red cell enzymes to gene dosage and gene expression determines the presence or absence of gene defects causing enzyme deficiencies leading to various metabolic diseases.
The mode of inheritance of these defects can frequently be ascertained by analysing red cell enzymes (ATPases, Protein Kinases, 3-phosphate dehydrogenase etc.).
Most red cell enzymes are tightly bound to the membrane and are present in small amounts.
Apparent enzyme deficiencies may result from the accumulation of inhibitory metabolites formed due to an enzyme deficiency in other tissues.

Learning Points

How do microarrays work in general?
What are the probes, and what are the labels?
How are the probes put on the arrays?
How do you determine the expression profile?
The rates of expression of every gene in a cell are simultaneously measured by DNA chips.

Key Components

Probes
- Nucleic Acids
- DNA
- cDNA
- Antibody
- Peptides
- Glycans
Microarray/Array/Chip
Detection System
- Nucleotide
- Monosaccharide
- Aminoacid
- Hybridization