1. Introduction to genome biology and microarrays experiment
Statistics for Microarray Data Analysis – Lecture 1
The Fields Institute for Research in
Mathematical Sciences
May 25, 2002

2. The human genome
The cell is the fundamental working unit of every living organism.
Humans: trillions of cells (metazoa);
other organisms like yeast: one cell (protozoa).
Cells are of many different types (e.g. blood, skin, nerve cells), but all can be traced back to a single cell, the fertilized egg.

3. Genes The human genome is distributed along 23 pairs of chromosomes.
22 autosomal pairs;
the sex chromosome pair, XX for females and XY for males.
In each pair, one chromosome is paternally inherited, the other maternally inherited.
Chromosomes are made of compressed and entwined DNA.
A (protein-coding) gene is a segment of chromosomal DNA that directs the synthesis of a protein.

4. Chromosomes and DNA

5. DNA
A deoxyribonucleic acid or DNA molecule is a double-stranded polymer composed of four basic molecular units called nucleotides.
Each nucleotide comprises a phosphate group, a deoxyribose sugar, and one of four nitrogen bases: adenine (A), guanine (G), cytosine (C), and thymine (T).
The two chains are held together by hydrogen bonds between nitrogen bases.
Base-pairing occurs according to the following rule: G pairs with C, and A pairs with T.

7. Genetic and physical maps

8. Genetic and physical maps
Physical distance: number of base pairs (bp).
Genetic distance: expected number of crossovers between two loci, per chromatid, per meiosis.
Measured in Morgans (M) or centiMorgans (cM).
1cM ~ 1 million bp (1Mb) in humans

9. Central dogma
The expression of the genetic information stored in the DNA molecule occurs in two stages:
(i) transcription, during which DNA is transcribed into mRNA;
(ii) translation, during which mRNA is translated to produce a protein.
DNA  mRNA  protein
Other important aspects of regulation: methylation, alternative splicing, etc.
The correspondence between DNA's four-letter alphabet and a protein's twenty-letter alphabet is specified by the genetic code, which relates nucleotide triplets to amino acids.

10. Idea: measure the amount of mRNA to see which genes are being expressed in (used by) the cell.
Measuring protein might be better, but is currently harder.

11. Differential expression
Each cell contains a complete copy of the organism's genome.
Cells are of many different types and states
E.g. blood, nerve, and skin cells, dividing cells, cancerous cells, etc.
What makes the cells different?
Differential gene expression, i.e., when, where, and in what quantity each gene is expressed.
On average, 40% of our genes are expressed at any given time.

12. RNA
A ribonucleic acid or RNA molecule is a nucleic acid similar to DNA, but
single-stranded;
ribose sugar rather than deoxyribose sugar;
uracil (U) replaces thymine (T) as one of the bases.
RNA plays an important role in protein synthesis and other chemical activities of the cell.
Several classes of RNA molecules, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and other small RNAs.

13. Exons and introns
Genes comprise only about 2% of the human genome; the rest consists of non-coding regions, whose functions may include providing chromosomal structural integrity and regulating when, where, and in what quantity proteins are made (regulatory regions).
The terms exon and intron refer to coding (translated into a protein) and non-coding DNA, respectively.

14. Functional genomics
The various genome projects have yielded the complete DNA sequences of many organisms. E.g. human, mouse, yeast, fruitfly, etc.
Human: 3 billion base-pairs, thousand genes.
Challenge: go from sequence to function, i.e., define the role of each gene and understand how the genome functions as a whole.

15. Introduction to microarrays

16. Gene expression assays
DNA microarrays rely on the hybridization properties of nucleic acids to monitor DNA or RNA abundance on a genomic scale in different types of cells.
The main types of gene expression assays:
High-density nylon membrane arrays;
Serial analysis of gene expression (SAGE);
Short oligonucleotide arrays (Affymetrix);
Long oligonucleotide arrays (Agilent);
Fibre optic arrays (Illumina);
cDNA arrays (Brown/Botstein)*.

17. Applications of microarrays
Measuring transcript abundance;
Genotyping;
Estimating DNA copy number;
Determining identity by descent;
Measuring mRNA decay rates;
Identifying protein binding sites;
Determining sub-cellular localization of gene products;
……..

19. The process Building the chip: RNA preparation: Hybing the chip:
PCR PURIFICATION and PREPARATION
MASSIVE PCR
PREPARING SLIDES
PRINTING
RNA preparation:
Hybing the chip:
CELL CULTURE AND HARVEST
POST PROCESSING
ARRAY HYBRIDIZATION
RNA ISOLATION
DATA ANALYSIS
cDNA PRODUCTION
PROBE LABELING
Taken from Schena & Davis

20. bacterial glycerol stocks)
Building the chip
PCR amplification
Directly from colonies with
SP6-T7 primers in 96-well
plates
Consolidate into
384-well plates
Arrayed Library
(96 or 384-well plates of
bacterial glycerol stocks)
Slide 4
Ideally, you will have a slides consist of all genes in the genomeThe design of the array / template is an imporatnt bioinformatics question. As you are only able to detect expression of genes on this template.
Spot as microarray
on glass slides
Ngai Lab, UC Berkeley)

21. 384 well plate Glass Slide cDNA clones Pins collect cDNA from wells
Contains cDNA probes
cDNA clones
Spotted in duplicate
Print-tip group 1
Glass Slide
Array of bound cDNA probes
4x4 blocks = 16 print-tip groups
Print-tip group 6

22. Sample preparation

23. Hybridization Binding of cDNA target samples to cDNA probes on the slide
cover
slip
Hybridize for
5-12 hours

24. Hybridization chamber
3XSSC
HYB CHAMBER
ARRAY
LIFTERSLIP
SLIDE
LABEL
SLIDE LABEL
Humidity
Temperature
Formamide
(Lowers the Tm)

25. Expression profiling with DNA 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

26. RGB overlay of Cy3 and Cy5 images

27. Raw data Human cDNA arrays ~ 43K spots;
16–bit TIFFs: ~ 20Mb per channel;
~ 2,000 x 5,500 pixels per image;
Spot separation: ~ 136um;
For a “typical” array:
Mean = 43, med = 32, SD = 26 pixels per spots

28. Image analysis
The raw data from a cDNA microarray experiment consist of pairs of image files, 16-bit TIFFs, one for each of the dyes.
Image analysis is required to extract measures of the red and green fluorescence intensities for each spot on the array.

29. Image analysis
GenePix

30. Image analysis R and G for each spot on the array.
1. Addressing. Estimate location of spot centers.
2. Segmentation. Classify pixels as foreground (signal) or background.
3. Information extraction. For
each spot on the array and each
dye
signal intensities;
background intensities;
quality measures.
Motivation behind background adjustment: A spot’s measured fluorescence intensity includes a contribution that is not specifically due to the hybridization of the target to the probe, but to something else, e.g. the chemical treatment of the slide, autofluorescence etc. Want to estimate and remove this unwanted contribution.
R and G for each spot on the array.

31. Spot Batch automatic addressing.
Segmentation. Seeded region growing (Adams & Bischof 1994): adaptive segmentation method, no restriction on the size or shape of the spots.
Intensity extraction
Foreground. Mean of pixel intensities within a spot.
Background. Morphological opening: non-linear filter which generates an image of the estimated background intensity for the entire slide.
Spot quality measures.
Software package. Spot, built on the freely available software package R.

32. Quality measures Spot One channel, R or G Signal/noise ratio;
Variation in pixel intensities;
Identification of “bad spots” (no signal), etc.
Two channels, R/G
Brightness: foreground/background ratio;
Uniformity: variation in pixel intensities and ratios of intensities;
Morphology: area, perimeter, circularity.
Array (slide)
Percentage of spots with no signal;
Range of intensities;
Distribution of spot signal area, etc.
How to use quality measures in subsequent analyses?

33. Terminology
Probe: DNA spotted on the array, aka. spot, immobile substrate.
Target: DNA hybridized to the array, mobile substrate.
Sector: collection of spots printed using the same print-tip (or pin), aka. print-tip-group, pin-group, spot matrix, grid.
Batch: collection of slides with the same probe layout.
The terms slide or array are often used to refer to the printed microarray.

34. Microarray Life Cycle Biological Question Data Analysis & Modelling
Sample Preparation
Microarray Life Cycle
MicroarrayDetection
Microarray Reaction
Taken from Schena & Davis

35. WWW resources Complete guide to “microarraying”
Parts and assembly instructions for printer and scanner;
Protocols for sample prep;
Software;
Forum, etc.
Animation:

36. Acknowledgments
Introduction to biology: based on Bioconductor short course lecture 1 ( and Temple short course lecture 1 with pictures from
Introduction to microarray: based on IPAM, UCLA
Thanks also to:
Natalie Thorne (WEHI)
Ingrid Lönnstedt (Uppsala)