Presented by: Akram Moslehi DNA Microarray Presented 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 How do we manufacture a spotted microarray? Spotted Glass Arrays Uses 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 “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

concentration of 100-500 µg/ml steel spotting pin Spotted arrays Spotting is done by a robot such as inkjet printing 384 well source plate chemically modified slides 1 nanolitre spots 90-120 µm diameter concentration of 100-500 µg/ml

Longer sequence target:500-2000bp 3.6cm2 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 genome Nonoverlapping

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 (1) isolating a total RNA containing m RNA (2)converted into (cDNA) (3)labelled with Cy3 , Cy5 (4)purified to remove contaminants such as primers, unincorporated nucleotides, cellular proteins, lipids, and carbohydrates filter spin columns :Qiaquick

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

(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. (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.

Expression profiling with cDNA microarrays cDNA “B” Cy3 labeled cDNA “A” Cy5 labeled Laser 1 Laser 2 Hybridization Scanning Slide 5 You will have another sample of interests consists of a portion of the genes and the questions is to find out what genes are being expressed in this sample. The simplified version of the experiments is to label the sample with a color dye hybridize the sample on the slides and scan the slides under some laser to see which spots that light up. This provides a list of genes and it’s corresponding expression levels in our samples. Our interest is on comparing the expression levels between the two samples. + Analysis Image Capture

Microarray confocal scanner

Image analysis of cDNA array

Expression profiling with DNA microarrays Data acquistion April 19, 2017 Expression profiling with DNA microarrays Data acquistion cDNA “control” Cy3 labeled 6 1 5 2 3 cDNA “treament” Cy5 labeled 1 6 5 3 4 Relative intensity HYBRIDIZATION Output 5 3 6 1 2 4 Spot #1 Spot #4 Acquistion=بدست آوری Spot #2 Spot #5 Spot #3 Spot #6

Reading an array (cont.) Block Column Row Gene Name Red Green Red:Green Ratio 1 tub1 2,345 2,467 0.95 2 tub2 3,589 2,158 1.66 3 sec1 4,109 1,469 2.80 4 sec2 1,500 0.42 5 sec3 1,246 1,258 0.99 6 act1 1,937 2,104 0.92 7 act2 2,561 1,562 1.64 8 fus1 2,962 3,012 0.98 9 idp2 3,585 1,209 2.97 10 idp1 2,796 1,005 2.78 11 idh1 2,170 4,245 0.51 12 idh2 1,896 2,996 0.63 13 erd1 1,023 3,354 0.31 14 erd2 1,698 2,896 0.59 Campbell & Heyer, 2003

Image Analysis & Data Visualization Cy3 Cy5 log2 Genes Experiments 8 4 2 fold 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.

Validating Microarray Expression Data 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

Microarray Validation Two 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 Medicine Clinical 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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