Introduction to Introduction to Bioinformatics:

Microarray Technology

Assc.Prof. Chuchart Areejitranusorn

AMS. KKU.

ความจริงเกี่ยวกับความจรงเกยวกบ Cell and DNADNA

Cell NucleusCell

Chromosome

ProteinGene (DNA)

Gene (mRNA), single strand

TranscriptionRNA polymerase

DNA RNA

atio

nReverse Transcription

RNA dependent

Tran

slaRNA dependent

DNA polymerase

T

PROTEIN

ิ ี่ ั C ll dความจริงเกียวกับ Cell and DNAAll living organisms consist of cells. Humans have trillions of cells; Yeast - one cell.cells; Yeast one cell.

Cells are of many differenttypes (blood skin nerve) but types (blood, skin, nerve), but all arose from a single cell (the fertilized egg)(the fertilized egg)

ความจริงเกีย่วกับ Cell and DNA

Each cell contains a complete Each cell contains a complete copy of the genome (the program for making the program for making the organism), encoded in DNA.

A gene is a segment of DNA g gthat specifies how to make a protein. Human DNA has about p30-35,000 genes

Gene Expression

•Cells are different because of differential gene expressiongene expression.

•About 40% of human out of human genes are expressed t tiat one time.

Gene Expression

•Gene is expressed by Gene is expressed by transcribing DNA into gsingle-stranded mRNA• RNA i l t t l t d•mRNA is later translatedinto a proteinp•Microarrays measure the l l f RNA ilevel of mRNA expression

Microarray DeviceMicroarray Device• A Microarray is a device

d h d y

detects the presence and abundance of labelled nucleic

d l l lacids in a biological sample.

• In the majority of experiments, the labelled nucleic acids are the labelled nucleic acids are derived from the mRNA of a sample or tissuesample or tissue.

Designing the Probes•high specificity to avoid hybridization with wrong target hybridization with wrong target molecules.••an output that is easy to read•high sensitivity to detect the high sensitivity to detect the mRNA and the intensity of the

t li ht t b spot light must be differentiable from background gnoise.

Designing the Probes

•The intensity of a spot light l d t l t ith also needs to correlate with

the abundance of the target the abundance of the target molecule in the sample.

•Results must be reproducible m lti l x im tacross multiple experiments.

cDNA Probe

• l •polymerase chain reaction ( ) (PCR) products (cDNAs)( )•amplified DNA is purifiedis purified,• the clones are typically long typically long sequences

Oligo probe(In-situ Synthesis Affymetrix)

The Array

A li l tid An oligonucleotide, or oligo as it is

l ll d commonly called, is a short f f fragment of a single-stranded DN h

gDNA that is typically 5 to 50 yp ynucleotides long.

The Operation of the Spotting Robot

The pins are dipped into the wells to collect the first b t h f DNAbatch of DNA.

This DNA is spotted onto a number of different arrays, depending on the y , p gnumber of arrays being made and the amount of liquid the pins can hold.

The pins are washed toThe pins are washed to remove any residual solution and ensure no contamination of the next samplesample.

The pins are dipped into the next set of wells.

Return to step 2 and repeat p puntil the array is complete.

Comparison of Probe Types

AdvantagesOligos probe cDNA Probes

AdvantagesAdvantages• No need to isolate and

purify cDNAs

Advantages• Flexibility to study

cDNAs from any source.purify cDNAs • Short oligonucleotides

are less likely to have

cDNAs from any source.• cDNAs do not require

any a priori information cross-reactivity with other sequences in the target DNA

about the corresponding genes.

• Longer sequences target DNA.• Density of chips is

higher than with cDNAs.

• Longer sequences increase hybridization specificity, which h gher than w th cDNAs. spec f c ty, wh ch reduces false positives.

Comparison of Probe Types

Limitations

Oligos probe cDNA Probes

LimitationsLimitations• The sequence has to be

known.

Limitations• Isolation of individual

cDNAs to immobilize on known.• Synthesis can be

expensive and time-

cDNAs to immobilize on each spot can be cumbersome.

consuming.• The short sequences are

not as specific for

• Density is lower • cDNAs are longer

sequences and are more not as specific for target DNA, so appropriate controls

sequences and are more likely to randomly contain sequences found appropriate controls

must be added.contain sequences found in target DNA, which results in cross-

Steps of a Microarray Experiment

choosing probes.

Generate a hybridization solution containing a mixture of

fluorescently labelled targetsfluorescently labelled targets.

Incubate hybridization mixture.y

Detect probe hybridization using laser technology laser technology

Analyze data using computational y g pmethods.

I l l l In Single label experiments, only one sample is hybridised to the arrays labelled with one dye. (in which case control needs to be measured using a separate chip)control needs to be measured using a separate chip).

Dual Label ExperimentspMost laboratories use fluorescent

labelling with the two dyes Cy3 labelling, with the two dyes Cy3 (excited by a green laser) and Cy5 (excited by a red laser) (excited by a red laser).

In Dual label experiments, two samples are hybridised to the arrays, one labelled with each dye; this allows the simultaneous measurement of two samples (e.g. for differential analysis)

Dual Label Experiments

+ Green label + Red label

RNA sample 1 RNA sample 2

Typically used to study one sample (e.g. diseased tissue) vs. a control sample (e.g. ) p ( gnormal tissue)

The Process

elled targets gin solution

Probes on array Heteroduplexes

Hybridisation

Qualitative Interpretation of Double Label ExperimentsDouble Label Experiments

GREEN = High Control hybridizationRED = High Sample hybridizationRED = High Sample hybridizationYELLOW = combination of Control and

l h b h h b d d Sample where both hybridized equally.qua y.

BLACK = neither the Control nor Sample hybridizedhybridized.

Spot Quality Problemsp y

From Microarray images to Gene Expression MatricesGene Expression Matrices

Final data

Images

Intermediate data

Samples

Final data Gene Expression Matrix

Raw data

ts nes

Array scans

Spot

Gen

GeneSpot/Image quantiations

Gene expression levels

PM to maximize hybridization MM to ascertain the degree of cross-hybridization

Affymetrix Gene ChipsPerfect Matches and MismatchesPerfect Matches and Mismatches

Microarray reader

Influenza strains

Commercial kits•SARS•Hepatitis•TBTB•Mycoplasma pneumonea• E t i i f ti Entero virus infection • Papilloma virus• Food•MilkMilk• Etc.

http://www affymetrixReferences

http://www.affymetrix.com/

http://www.gene-chip comchip.com

http://www.icscience.cสวัสดี p //om

สวสด

http://www.csus.edu