An introduction to Microarrays

Dr. Vipin Singh, Amity University, Noida

WHAT IS MICROARRAY
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
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.

Probes
Features 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) cDNA probes 500- 5000 bases long, single stranded cDNA immobilized on chip

Probes are immobilized on to the chip by one of the following three methods -Inkjet printing -Photolithography -Robotic spotting

Affymetrix GeneChips
Oligonucleotide Chips
Usually 2025 bases in length 1020 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
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.

(12-20/gene)

probe pair

Mismatch probe cells

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.

SINGLE CHANNEL

MULTI CHANNEL

cDNA

cDNA

Non differential labeling Radiolabelling or flourochrome Competitive hybridization

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

Features of microarray
Miniaturization Small chip size

Parallelism Thousands of genes simultaneously

Multiplexing Multiple samples at the same time Automation Chip manufacturing Software aided analysis of results

A typical Microarray result (Single Channel Chip)

a) Patient

b) Control (Normal)

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.

Uses of microarrays Gene discovery - tissue profiles - time course data - altered genetic backgrounds Comparing tissues/genotypes there are still some inherent difficulties here Classification theres a lot of promise in medicine (especially cancer research) for this

Experimental artifacts in microarray

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

Overcoming 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

Technical vs biological replicates

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

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. Its not clear to what degree this affects results, especially with respect to rare transcripts, but seems to be generally OK if done correctly

Dye swap
When using 2-color arrays, its 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 S1

S2 S2

Controls
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

compare S1/R vs. S2/R vs. S3/R

Data Normalization
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.

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. Its usually too late and too expensive to change the design once youve begun!

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: 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