Microarray Technology

Microarrays And Functional Genomics CPSC265 Matt Hudson

Microarray Technology

• Usually used like a Northern blot – can determine the amount of mRNA for a particular gene

• Except – a Northern blot measures one gene at a time

• A microarray can measure every gene in the genome, simultaneously

Recent! History

• 1994. First microarrays developed by Ron Davis and Pat Brown at Stanford.

• 1997-1999. Practical microarrays become available for yeast, humans and plants

Why analyze so many genes?

• Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function. • To find genes involved in a particular process, we can

look for mRNAs “up-regulated” during that process. • For example, we can look at genes up-regulated in

human cells in response to cancer-causing mutations, or look at genes in a crop plant responding to drought. • Patterns/clusters of expression are more predictive

than looking at one or two prognostic markers – can figure out new pathways

Two Main Types of Microarray

Oligonucleotide, photolithographic arrays “Gene Chips”

Miniaturized, high density arrays of oligos (Affymetrix Inc., Nimblegen, Inc.)

Printed cDNA or Oligonucleotide Arrays

•Robotically spotted cDNAs or Oligonucleotides • Printed on Nylon, Plastic or Glass surface • Can be made in any lab with a robot • Several robots in ERML

• Can also buy printed arrays commercially

The original idea

A microarray of

thousands of

genes on a

glass slide

Each “spot” is one gene, like a probe in a Northern blot.

This time, the probes are fixed, and the target genes move about.

Glass slide microarray summary

The process

MASSIVE PCR PCR PURIFICATION and PREPARATION

PREPARING SLIDES PRINTING

RNA preparation: CELL CULTURE AND HARVEST RNA ISOLATION cDNA PRODUCTION Hybing the

chip: POST PROCESSING

ARRAY HYBRIDIZATION

PROBE LABELING

DATA ANALYSIS

Robotically printed arrays

Robotically printed arrays

1 nanolitre spots 90-120 um diameter

384 well source plate chemically modified slides steel

Physical Spotting

Reverse Transcriptase

Labelling RNA for Glass slides

mRNA (control) cDNA Cy3 labelled Reverse transcription mRNA (treated) cDNA Cy5 labelled Cy3 label Cy5 label

Hybridization

Binding of cDNA target samples to cDNA probes on the slide

cover slip

Hybridize for 5-12 hours

Northern blot vs. Microarray

• In Northern blotting, the whole mRNA of the organism is on the membrane. The labelled “probe” lights up a band – one gene

• In a microarray, the whole genome is printed on a slide, one “probe” spot per gene. Mixed, labelled cDNA, made from mRNA from the organism, is added. Each probe lights up green or red according to whether it is more or less abundant between the control and the treated mRNA. LABEL 3XSSC HYB CHAMBER ARRAY SLIDE LIFTERSLIP SLIDE LABEL • Humidity • Temperature • Formamide (Lowers the Tm)

Hybridization chamber

Expression profiling with DNA microarrays

Analysis Image Capture

Image analysis

GenePix

Spotted cDNA microarrays

• Lower price and flexibility

• Can be printed in well equipped lab

• Simultaneous comparison of two related

biological samples (tumor versus normal, treated versus untreated cells)

Disadvantages

• Needs sequence verification

• Measures the relative level of expression between 2 samples

Affymetrix Microarrays

…unlike Glass Slide Microarrays

•Made by a spotting robot •~30,000 50-500 base probes •Involves two dyes/one chip •Control and experiment on same chip

Affymetrix GeneChip

Miniaturized, high density arrays of oligos 1.28-cm by 1.28-cm (409,000 oligos) Manufacturing Process

Solid-phase chemical synthesis and Photolithographic fabrication techniques employed in semiconductor industry

Selection of Expression Probes

Set of oligos to be synthesized is defined, based on its ability to hybridize to the target genes of interest

Probes Sequence

Perfect Match

Mismatch Chip

5’ 3’

Computer algorithms are used to design photolithographic masks for use in manufacturing

Photolithographic Synthesis

Manufacturing Process

Probe arrays are manufactured by light-directed chemical synthesis process which enables the synthesis of hundreds of thousands of discrete compounds in precise locations

Lamp

Mask Chip

Affymetrix Wafer and Chip Format

1.28cm 50… 11µm 20 - 50 µm Millions of identical oligonucleotides per feature 49 - 400 chips/wafer up to ~ 400,000 “features” / chip

Reverse Transcriptase

in vitro transcription

Labelling RNA for Affymetrix

mRNA cDNA Reverse transcription Transcription Biotin labelled nucleotides cRNA

Target Preparation

Wash & Stain

Scan Hybridize (16 hours) mRNA AAAA B B B B Biotin-labeled transcripts Fragment (heat, Mg2+₎ Fragmented cRNA B _B B B

GeneChip

Expression Analysis

Hybridization and Staining

Array cRNA Target Hybridized Array Streptravidin-phycoerythrin conjugate

Example: Comparing a mutant cell line with a wild type line.

Instrumentation

3000-7G Scanner 450 Fluidic Station

Microarray data analysis

This is now a very important branch of statistics

It is unusual to do thousands of experiments at once. Statistical methods didn’t exist to analyse microarrays. Now they are being rapidly developed.

Normal vs. Normal

Normal vs. Tumor

Lung Tumor:

Up-Regulated

Lung Tumor:

Down-Regulated

Microarray Technology - Applications

• Gene

Discovery-–Assigning function to sequence

–Finding genes involved in a particular process

–Discovery of disease genes and drug targets • Genotyping

–SNPs

–Genetic mapping (Humans, plants)

–Patient stratification (pharmacogenomics)

–Adverse drug effects (ADE) • Microbial ID

What DNA microarrays can’t do

• Tell you anything about protein levels

• Tell you anything about post-translational modification of proteins

• Tell you anything about the structure of proteins

• Predict the phenotype of a genetic mutant

Proteomics

• A high througput approach to learning about all the proteins in a cell

• As microarrays are to a Northern blot, proteomics is to a Western blot

• Two main approaches –

• 2D gels + MS

• Protein microarrays

Protein separation:

2-dimensional gel electrophoresis 1st dimension

Separation by charge (isoelectric focussing)

2nd dimension

Separation by molecular weight (SDS-PAGE)

kDa

pH 3 pH 10

pI

Proteins extracted from cow ovarian follicle granulosa cells separated on a broad range IPG strip (pH3-10) followed by a 12.5% polyacrylamide gel, silver stained

3.5 9.0 20 150 100 75 50 37 25 Susan Liddel

Mass Spectrometry

FT-MS can tell you 10-20 residues of sequence, but only from a purified protein Robots pick spots from 2-D gel, load into MS Also, 2-D and 3-D LC

Protein microarrays

The future of microarrays:

•Still looking good

•Used by most pharmaceutical companies, almost all University biology departments •In the future, just like silicon chips, likely

to get cheaper, faster and more powerful •It may not be long before they are routinely

used to diagnose disease

The future of proteomics:

• Many people will tell you proteomics IS the future of biology

• If they can get it to work as well as microarrays, they will be right

• The problem is, every protein has different chemistry, while all mRNAs are closely comparable

• At the moment, proteomics is a hot field, but few major biological discoveries have been made with proteomics – many have been made with microarrays