Microarrays,RNAseq AndFunctionalGenomics

CPSC265

Matt Hudson

Microarray Technology• Relatively young technology – • Already mostly obsolete, though.

• 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 processBuilding the chip:

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 arraysRobotically printed arrays

1 nanolitre spots90-120 um diameter

384 well source plate

chemically modified slides

steel

spotting pin

Physical Spotting

Reverse Transcriptase

Labelling RNA for Glass slides

mRNA(control)

cDNACy3 labelled

Reverse transcription

mRNA(treated)

cDNACy5 labelled

Cy3 label

Cy5 label

HybridizationBinding 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

cDNA “A ”Cy5 labeled

cDNA “B”Cy3 labeled

Hybridization Scanning

Laser 1 Laser 2

+

Analysis Image Capture

Image analysis

GenePix

Spotted cDNA microarraysAdvantages• 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

• One chip per sample• Made by photolithography• ~500,000 25 base probes

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

Probes

Sequence

Perfect Match

MismatchChip

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

TranscriptionBiotin labellednucleotidescRNA

Target Preparation

cDNA

Wash & Stain

Scan

Hybridize

(16 hours)

mRNAAAAA

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-phycoerythrinconjugate

Example:

Comparing a mutant cellline with awild typeline.

Instrumentation

Affymetrix GeneChip System3000-7G 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

Why it is becoming obsolete

• In a word, RNAseq

• RNAseq uses DNA sequencing to do the same thing.

• Rather than an array, you just sequence millions of mRNA fragments, then figure out what genes they are from

Why RNAseq only just caught on

• It’s been around for a long time, called things like SAGE and MPSS.

• But they were expensive and arrays were cheap. Now, sequencing is as cheap as arrays

• Also, you need a fully sequenced reference genome for the computer analysis.

What RNAseq / arrays 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

Susan Liddel

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 you10-20 residues ofsequence, but onlyfrom a purified protein

Robots pick spots from2-D gel, load into MS

Also, 2-D and 3-D LC

Array-based protein interaction detection

Protein microarrays

The future of microarrays:

•Still looking good, in areas other than research

•Used by pharmaceutical companies, medical diagnostics, etc.

•In the future, just like silicon chips, likely to get cheaper, faster and more powerful

•It may not be long before they are routinelyused 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