An introduction to Microarrays

Dr. Vipin Singh,Amity University, Noida

Microarray is a nucleic acid hybridization based, high throughput

technique developed to quantitate gene expression levels at the

whole genome scale

Principle: base-pairing hybridization

– Central platform for functional genomics

WHAT IS MICROARRAY

Functional Genomics – Refers to genome wide analysis of gene function in contrast to studying individual genes or proteins

BASIC DESIGN OF MICROARRAY CHIP

Microarray chip basically represents a square or rectangular solid support , further divided into smaller subsquares,

Each of these subsquares is enriched with multiple probes (single stranded oligonucleotides) of a specific known gene.

ProbesFeatures immobilized on the chip surface are referred to as probes – may be of two

types –

Oligonucleotide probes - size ranges from 25 bases (as in Affymetrix chips) to 60 to 80

bases (in other companies)

Probes are immobilized on to the chip by

one of the following three methods

-Inkjet printing

-Photolithography

-Robotic spotting

cDNA probes – 500- 5000 bases long, single stranded cDNA immobilized on chip

Affymetrix GeneChips

• Oligonucleotide Chips– Usually 20–25 bases in length– 10–20 different oligonucleotides for each gene

• Oligonucleotides for each gene selected by computer program to be the following:– Unique in genome– Nonoverlapping

• Composition based on design rules• Empirically derived

Affymetrix GeneChips

probe pair Mismatch probe cells

(12-20/gene)

Each gene is represented by a “probe set” consisting of 12-20 probes of 25 nt each.

Each probe has a corresponding “mismatch” probe with a single base difference at the 13th nucleotide.

Labeled RNA is hybridized to the array, and a measure of abundance is calculated based on the amount of hybridization seen for the entire probe set, correcting for hybridization to the mismatch probes, which indicates possible non-specific effects.

Target

The time and stage specific RNA pool whose expression levels are to be quantified. The

RNA pool is labeled either with radio activity or with flourochromes

Two types of chips can be distinguished

1.Single Channel chip – each chip can hybridize only one type of target e.g. either

Normal or Cancer cell m RNA pool e.g. Cloneech or Affymetryx chips

2.Multichannel chip – two types of targets can be hybridized on the same chip eg.

Normal and Cancer m RNA pool (provided the two targets are differentially labelled –

normal with Cy3 and cancer with Cy5 or vice versa) eg. NEN life science arrays

Target labeling - Cyanins

synthetic dye family belonging to polymethine group

Cy 3 and Cy5 are the most popular Cyanine dyes, used typically combined for 2 color detection.

Cy 3 – Absorbance max – 550nm, emission max – 570nm

Cy5 – Absorbance max – 649nm, emission max – 670 nm

In microarray experiments DNA or RNA is labeled with either Cy3 or Cy5 that has been synthesized to carry an N-hydroxysuccinimidyl ester (NHS-ester) reactive group. Since, NHS-esters react readily only with aliphatic amine groups, which nucleic acids lack, nucleotides have to be modified with aminoallyl groups. This is done through incorporating aminoallyl-modified nucleotides during synthesis reactions. A good ratio is a label every 60 bases such that the labels are not too close to each other, thus resulting in quenching effects.

cDNA cDNA

Non differential labeling – Radiolabelling or flourochrome

Hybridize to one chip

Hybridize to another chip

Compare corresponding spots for relative quantification Compare red and green intensity of

each spot for relative quantification

SINGLE CHANNEL MULTI CHANNEL

Competitive hybridization

SINGLE CHANNEL

MULTI CHANNEL

– Miniaturization• Small chip size

– Parallelism• Thousands of genes simultaneously

– Multiplexing• Multiple samples at the same time

– Automation• Chip manufacturing• Software aided analysis of results

Features of microarray

Example of a microarray experiment using radioactive probes: 588 genes are represented on each array and are spotted in adjacent pairs. Dark dots represent genes expressed at high levels. The filters were hybridized, washed, and exposed to a phosphorimager screen for 6 hours. The output includes a quantitation (in pixel units) of the signals. (a) Clontech Atlas Neurobiology array probed with cDNA derived from the postmortem brain of a girl with Rett syndrome, and (b) the profile from a matched control. The arrows point at an RNA transcript (bcrystallin) that is upregulated in the disease. Note that overall the RNA transcript profiles appear similar in the two brains.

A typical Microarray result (Single Channel Chip)

a) Patient b) Control (Normal)

Uses of microarrays

• Gene discovery- tissue profiles- time course data- altered genetic backgrounds

• Comparing tissues/genotypes

there are still some inherent difficulties here

• Classification

there’s a lot of promise in medicine (especially cancer research) for this

Different labeling efficiencies of fluorescently (or radioactively) labeled nucleotides

Technical artifacts such as uneven spotting of DNA onto the array surface

Variations in the performance of a fluorescence scanner or phosphorimager

Variations in the RNA (or mRNA) purity or quantity among the biological samples being studied

Variations in the way the RNA is purified, labeled, and hybridized to the microarray

Variations in the way washing is done to remove nonspecific binding

Variations in the way the signal is measured

Experimental artifacts in microarray

There are a number of important experimental design considerations for a microarray experiment

• technical vs biological replicates

• amplification of RNA

• dye swaps

• reference samples

• Data normalization

Overcoming artifacts in microarray

•technical replicates are repeat hybridizations using the same RNA isolate

•biological replicates use RNA isolated from separate experiments/experimental organisms

Technical replicates can be useful for reducing variation due to hybridization, imaging, etc.,

biological replicates are necessary for a properly controlled experiment

Technical vs biological replicates

Experimental Design for Microarrays

Amplification of RNA

• linear amplification methods can be used to increase the amount of RNA so that microarray experiments can be performed using very small numbers of cells. It’s not clear to what degree this affects results, especially with respect to rare transcripts, but seems to be generally OK if done correctly

When using 2-color arrays, it’s important to hybridize replicates using a dye-swap strategy in which the colors (labels) are reversed between the two replicates.

This is because there can be biases in hybridization intensity due to which dye is used (even when the sequence is the same).

S1 S2

S1 S2

Dye swap

Reference samples

•one common strategy is to use a reference sample in one channel on each array. This is usually something that will hybridize to most of the features (e.g., a complex RNA mixture). Using a reference sample allows comparisons to be made between different experimental conditions, as each is compared to the common reference.

S1

S2

S3

R

R

R

compareS1/R vs. S2/R vs. S3/R

Controls

choose a gene-set (constitutive genes - expression levels do not change under the conditions studied) expected to have an expression ration of 1.

From this set, a normalization factor, which is a number that accounts for the variability seen in the geneset, is calculated. It is then applied to the other genes in the microarray experiment.

One should note that the normalization procedure changes the data, and is carried out only on the background corrected values for each spot.

Data Normalization

Experimental Design for Microarrays

The bottom line is that you should discuss your

experimental design with a statistician before going ahead

and beginning your experiments. It’s usually too late and

too expensive to change the design once you’ve begun!

• EXPERIMENT DESIGN - type, factors, number of arrays, reference sample, qc, database accession (ArrayExpress, GEO)

• SAMPLES USED, PREPARATION AND LABELING

• HYBRIDIZATION PROCEDURES AND PARAMETERS

• MEASUREMENT DATA AND SPECIFICATIONS - quantitations, hardware & software used for scanning and analysis, raw measurements, data selection and transformation procedures, final expression data

• ARRAY DESIGN - platform type, features and locations, manufacturing protocols or commercial p/n

MIAME (Minimal Information About a Microarray Experiment)

When you publish a microarray experiment, you are expected to make available the following minimal information. This allows others to evaluate your data and compare it to other experimental results: