DNA MicroarrayPresented by: Akram Moslehi

High-throughput methods for measuring cellular states• Gene expression levels: RT-PCR, arrays

• Protein levels, modifications: mass spec • Protein locations: fluorescent tagging

• Metabolite levels: NMR and mass spec

• Systematic phenotyping

Outline of the lecture

Overview of Microarray Technology Applications

Types of Microarrays Manufacturing

Instrumentation and Software Data Analysis-Basic

What are Microarrays?

• Microarrays are simply small glass or silicon slides upon the surface of which are arrayed thousands of features (usually between 500 up to a million)

• Using a conventional hybridization process, the level of expression of genes is measured (for instance)

• Microarrays are read using laser-based fluorescence scanners

• The process is “high throughput”

Why use Microarrays?•Determine what genes are active in a cell and at what levels

• Compare the gene expression profiles of a control vs treated

• Determine what genes have increased or decreased in during an experimental condition

• Determine which genes have biological significance in a system

• Discovery of new genes, pathways, and cellular trafficking

Types of microarrays

• Spotted (cDNA) - by Patrick Brown in 1990– Robotic transfer of cDNA clones or PCR products– Spotting on nylon membranes or glass slides coated with poly-lysine– ink-jet printing (Agilent)

• Synthetic (oligo) – by Stephen Fodor in 1991– Direct oligo synthesis on solid microarray substrate– Uses photolithography (Affymetrix) or ink-jet printing (Agilent)

• Labeling can be radioactive, fluorescent (one-color), or two-color

How do we manufacture a spotted microarray?

Spotted Glass ArraysUses cDNA, Oligonucleotide, protein, antibody

-Robotically spotted cDNAs or Oligonucleotides -Printed on Nylon, Plastic, or Glass microscope slide

Agilent: Oligonucleotide Array

Glass slides characteristics

• excellent chemical resistance against solvents,

• good mechanical stability (increased thermal strain point)

• low intrinsic fluorescence properties.

Start with individual genes

Amplify

purity by sequencing or using on agarose gel and an estimate of the DNA concentration

This is an important step because all the DNA fragments should be of similar concentration/molarity and size, to achieve similar reaction kinetics for all hybridisations

“Spot” them on a medium, e.g. an ordinary glass microscope slide that chemically modified glass slides usually with poly(L-lysine) or other cross-linking chemical coating materials such as polyethyleneimine polymer p-aminophenyl trimethoxysilane/diazotization

the DNA solution will be immobilised on the surface e.g. covalent or non covalent. However in the course of poly (L-lysine) the negatively charged phosphate groups in the DNA molecule, form an ionic bond with the positively charged amine-derivatised surface

Spotted arrays

384 well source plate

chemically modified slides1 nanolitre spots

90-120 µm diameter

concentration of 100-500 µg/ml

steel

spotting pin

Spotting is done by a robot such as inkjet printing

3.6cm 2

200-250µm

Longer sequence target:500-2000bp

Microarray Spotter

Robotic spotting

The post-print processing step :drying of the DNA on the slide overnight at room temperature and the use of UV cross-linking to prevent subsequent binding of DNA, and to decrease the background signal upon hybridisation

of a labelled target

Cartridge-based Chips

-Miniaturized, high density arrays of DNA oligos within a plastic housing

-One sample=One chip (Affymetrix, Agilent, Applied Biosystems…)

-Uses single fluorescent dye

-More expensive

-Usually 20–25 bases in length

-10–20 different oligonucleotides for each gene

500,000 Probes

GeneChip Technology Affymetrix Inc

• Miniaturized, high density arrays of 1,300,000 DNA oligos 1-cm by 1-cm

• Manufacturing Process:

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

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

Spotted Vs. Oligonucleotide array

Spotted Arrays Relative cheap to make

(~$10 slide) Flexible - spot anything

you want (2Kbp) Cheap so can repeat

experiments many times Highly variable spot

deposition Usually have to make your

own Cross hybridisation

Affy Gene Chips Expensive ($500 or

more) Limited types avail, no

chance of specialized chips

Fewer repeated experiments usually

Increases specificity, Decrease sensitivity

Can buy off the shelf

Sample preparation and labelling

filter spin columns :Qiaquick

(3)labelled with Cy3 , Cy5

(2)converted into (cDNA)(1) isolating a total RNA containing m RNA

(4)purified to remove contaminants such as primers,

unincorporated nucleotides, cellular proteins, lipids, and

carbohydrates

Sample preparation and labelling

• the sample preparation starts by isolating a total RNA containing messenger RNA that ideally represents a quantitative copy of genes expressed at the time of sample collection (experimental sample & reference sample).

• separately converted into complementary DNA (cDNA)

• each cDNA (Sample and Control) are labelled with a different tracking molecule, often fluorescent cyanine dyes (i.e. Cy3 and Cy5)

Array hybridisation

• the labelled cDNA (Sample and Control) are mixed together

• purified to remove contaminants such as primers, unincorporated nucleotides, cellular proteins, lipids, and carbohydrates. Purification is usually carried out using filter spin columns such as Qiaquick from Qiagen

• the mixed labelled cDNA is competitively hybridised against denatured PCR product or cDNA molecules spotted on a glass slide

(6)The slides are washed after hybridisation, first to remove any labelled cDNA that did not hybridise on the array, and secondly to increase stringency of the experiment to reduce cross hybridisation. The later is

achieved by either increasing the temperature or lowering the ionic strength of the buffers.

(5) Before hybridisation, the microarray slides are incubated at high temperature

with solutions of saline-sodium buffer (SSC), Sodium Dodecyl Sulfate (SDS)

and bovine serum albumin (BSA) to reduce background due to nonspecific

binding.

Expression profiling with cDNA microarrays

cDNA “A”Cy5 labeled

cDNA “B”Cy3 labeled

Hybridization Scanning

Laser 1 Laser 2

+

Analysis Image Capture

Microarrayconfocal scanner

Image analysis of cDNA array

33

Expression profiling with DNA microarrays Data acquistion

HYBRIDIZATION

Spot #1

Spot #3

Spot #2

Spot #4

Spot #6

Spot #5

Relative intensity

Relative intensity

cDNA “control”Cy3 labeled

6

1

555

5

2

2

2

2 33

Output Output

cDNA “treament”Cy5 labeled

1

6

6

6

5 55

5

3 6

6

4

5

3 6

666

66

11

55

5

55

552

2 2

2

33

4

34

Reading an array (cont.)Block Column Row Gene Name Red Green Red:Green

Ratio

1 1 1 tub1 2,345 2,467 0.95

1 1 2 tub2 3,589 2,158 1.66

1 1 3 sec1 4,109 1,469 2.80

1 1 4 sec2 1,500 3,589 0.42

1 1 5 sec3 1,246 1,258 0.99

1 1 6 act1 1,937 2,104 0.92

1 1 7 act2 2,561 1,562 1.64

1 1 8 fus1 2,962 3,012 0.98

1 1 9 idp2 3,585 1,209 2.97

1 1 10 idp1 2,796 1,005 2.78

1 1 11 idh1 2,170 4,245 0.51

1 1 12 idh2 1,896 2,996 0.63

1 1 13 erd1 1,023 3,354 0.31

1 1 14 erd2 1,698 2,896 0.59Campbell & Heyer, 2003

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Image Analysis & Data Visualization

200 10000 50.00 5.644800 4800 1.00 0.009000 300 0.03 -4.91

Cy3 Cy5Cy5Cy3

Cy5Cy3log2

Gen

es

Experiments 842

fold248

Underexpressed

Overexpressed

Normal vs. Normal Normal vs. Tumor

All configurations assume the DNA on the array is in excess of the hybridized sample, thus the kinetics are linear and the spot intensity reflects that amount of hybridized sample.

Microarray data are not stand alone results and requires validation by second method

Microarray data is only semi-quantitative because of a limited dynamic range.

True quantitative results must be determined with another technique such as Quantitative real-time PCR

Validating Microarray Expression Data

Microarray ValidationTwo types of validation

1] Validating the instrument data using the same RNA (confirming a result)

And most importantly

2] Validating the biological phenomenon with new samples same experiment conditions

Methods

Northern Blots, Immunohistochemistry, Western Blot,

PCR- i.e.Quantitative real-time PCR

**DNA mapping Arrays or CGH may also help indicate where or why a change is occuring

Microarray Future

Diagnostics -[Affy, Nanogene only at this time]– Disease detection– Tumor classification– Patient stratification– Intervention therapeutics

Treatment and Customized MedicineClinical arrays currently available are the AmpliChip CYP450 by Affymetrix and Roche. Used for predictive phenotyping in defects of the cytochrome P450 Genes

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