1. Data Type 1: Microarrays
Reverse Genetics approach
Genomics
So we need to understand what exactly is a Microarray
DNA Microarrays are small, solid supports(the size of two side-by-side pinky fingers) onto which the sequences from thousands of different genes are immobilized, or attached, at fixed locations.
So we need to KNOW the sequence to design this array.

2. Definition of Microarray
A semiconductor device that is used to detect the DNA makeup of a cell.
It contains hundreds of thousands of tiny squares designed to mate with a particular gene.
They react to the liquified cells poured over it and are detectable by a laser.
Microarrays are helping revolutionize medicine by being able to pinpoint a very specific disease or the susceptibility to it.
Sometimes called "biochips," micro arrays are commonly known as "gene chips," GeneChip is an Affymetrix trademark.

3. Microarrays have and will continue to do so.
revealed new patterns of coordinated gene expression across gene families,
expanded the size of existing gene families,
increased our understanding of how these genes coordinate
precise knowledge of these inter-relationships has emerged
speeded the identification of genes involved in the development of various diseases
aided the examination of the integration of gene expression and function at the cell level,
revealed how multiple gene products work together
and will continue to do so.

4. A Microarray

5. Gene Expression Analysis
Typical Northern Blot: One gene/ experiment/ more than one sample
Fairly quantitative
Time consuming
Limited information
Microarray and RNA-seq: thousands of gene/one sample
Less time
Massive information

6. Central dogma of molecular biology
REMEMBER:
Central dogma of molecular biology
Each gene is transcribed (at the appropriate time) from DNA into mRNA, which then leaves the nucleus and is translated into the required protein.
This is the principle used for microarrays

7. Types of Microarrays
Two channel spotted arrays: (more historical than used now)
cDNA microarrays: probes are cDNA base pairs in length, PCRd from custom libraries and spotted onto a microscope slide using a robot.
Long-oligo spotted arrays: shorter but uniform length nucleotide probes, bp, usually synthesized and spotted as with cDNA, but there are also
Commercial spotted arrays (e.g., Agilent) spotting longoligo probes using inkjet technology.
Single channel arrays:
High-density short (e.g. 25 bp) oligo arrays (Affymetrix,Nimblegen) synthesized in situ. Single-channel.
Some commercial arrays (e.g., Applied Biosystems AB1700)
Some Spotted Single Channel cDNA arrays (not that common but exists)

8. Single Channel AFFY chip

9. Affymetrix “Gene chip” system in 2007
Uses 25 base oligos synthesized in place on a chip (20 pairs of oligos for each gene)
RNA labeled and scanned in a single “color”
one sample per chip
Can have as many as 50,000 genes on a chip and it keeps increasing
Arrays get smaller every year (more genes)
Chips are expensive
Proprietary system: “black box” software, can only use their chips
TOUTED AS THE CHIP WITHOUT ISSUES

10. Current Affymetrix
Many products with lot more options for custom arrays.
Have products that can handle multiple samples at once.
Description for Affymetrix HG-U133, MG-430, and RG-230 Array
These data sources are used to design probes that interrogate 9 to 11 unique sequences of each transcript.
The unique 25-mer probes interrogate up to 275 bases per transcript.

11. Affy-GWAS chips
The new Affymetrix Genome-Wide Human SNP Array 6.0 features 1.8 million genetic markers, including more than 906,600 single nucleotide polymorphisms (SNPs) and more than 946,000 probes for the detection of copy number variation.
The high price-performance value of the SNP Array 6.0 enables researchers to design association studies with larger sample sizes in the initial scan and replication phases, thereby significantly increasing the overall genetic power of their studies.

12. Current Content
RefSeq probe sets HG-U133 HG-U219 MG-430 RG-230
NM – RefSeq coding transcript, 38,026 43,134 34,325 17,277
XM – RefSeq coding transcript, 1, ,021 2,692
NR – RefSeq non-coding transcript, 1,
XR – RefSeq non-coding transcript,
RefSeq probe sets (total) 41,090 43,932 35,892 20,208
UniGene probe sets 10,377 5,388 2,819 8,842
Other probe sets 3, ,412 2,070
Total probe sets 54,700 49,411 45,123 31,120

14. Probe Design: Affymetrix

15. Photolitigraphic Synthesis

16. Scanning AFFY chips
Light removes protecting groups at defined positions.
Single nucleotide washed over the chip, binds where the protecting group removed.
Through successive steps, any sequence can be built up in any position on the chip.
The number of steps corresponds with length of oligo, so can increase # of genes without # of steps

17. Analysis of expression level from probe sets.
Each pixel is quantitated and integrated for each oligo feature (range 0-25,000)
Perfect Match (PM)
Mis Match (MM) Control
PM - MM = difference score per probe set
All significant difference scores are averaged to create “average difference” = expression level of the gene.

18. Microarray Data Analysis
How to Handle Microarray Data?
Preprocessing:
Signal Generation from Image
• Normalization
• Filtering
Analysis:
• Statistical Tests for differential Expressions, t tests, non-parametric
Tests, ANOVA
• Clustering: Hierarchical, non-hierarchichal, SOM
Classification: Discriminant Analysis, PCA
The Main Goal of Microarray Data Analysis is to Generate a
List Of ‘Interesting’ Genes