3. Presented by: Akram Moslehi
DNA Microarray
Presented by: Akram Moslehi

4. 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

5. Outline of the lecture Overview of Microarray Technology Applications Types of Microarrays Manufacturing Instrumentation and Software Data Analysis-Basic

6. 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”

8. 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

9. 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

10. 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

11. Glass slides characteristics
excellent chemical resistance
against solvents,
good mechanical stability (increased thermal strain point)
low intrinsic fluorescence properties.

12. Start with individual genes
Amplify

13. 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

14. “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

15. 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
µm diameter
concentration of µg/ml

16. Longer sequence target:500-2000bp
3.6cm2 µm
Longer sequence target: bp

17. Microarray Spotter

18. Robotic spotting

19. 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

20. 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

21. 500,000 Probes

22. 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

24. 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

25. 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

26. 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)

27. 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

28. (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.

29. Expression profiling with cDNA microarrays
cDNA “B”
Cy3 labeled
cDNA “A”
Cy5 labeled
Laser 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

31. Microarray confocal scanner

32. Image analysis of cDNA array

33. 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

34. 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

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

36. 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.

37. 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

38. 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

39. 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

40. برگ در انتهای زوال می افتد و میوه در انتهای کمال پس بنگر چگونه میافتی چون برگی زرد و یا چون سیبی سرخ