Transcript 3/27

Genomics I:
The Transcriptome
RNA Expression Analysis
Determining genomewide RNA
expression levels
Genomewide expression
analysis
• Goal: to measure RNA levels of all genes
in genome
• RNA levels vary with the following:
– Cell type
– Developmental stage
– External stimuli
• Time and location of expression provide
useful information as to gene function
Genomics expression analysis
methods
• Microarrays
– Hybridization based
• RNA-seq
– Direct sequencing of cDNAs
• SAGE (Serial Analysis of Gene
Expression)
– Sequence fragments of cDNAs
• Real-time PCR
Macroarray Analysis
Macroarray Analysis
Microarray Analysis of Transcription
Animation
Northern blots vs. microarrays
target –
• Global expression
analysis: Northern
blot
– Limited by number of
probes that can be
used simultaneously
• Global expression
analysis: microarrays
– RNA levels of every
gene in the genome
analyzed in parallel
loading –
control
Basics of microarrays
• DNA attached to solid
support
– Glass, plastic, or nylon
• RNA is labeled
– Usually indirectly
• Bound DNA is the
probe
– Labeled RNA is the
“target”
Microarray hybridization
samples
• Usually comparative
– Ratio between two
samples
• Examples
– Tumor vs. normal
tissue
– Drug treatment vs. no
treatment
– Embryo vs. adult
mRNA
cDNA
DNA
microarray
Two major types of microarrays
• cDNA arrays- PCR product corresponding
to a portion of a cDNA is immobilized on
the slide
• oligonucleotide arrays- oligonucleotide
complementary to transcript is synthesized
on slide or immobilized on the slide
How microarrays are made:
spotted microarrays
• DNA mechanically placed on glass slide
• Need to deliver nanoliter to picoliter volumes
– Too small for normal pipetting devices
• Robot “prints,” or “spots,” DNA in specific places
DNA spotting I
• DNA spotting usually
uses multiple pins
• DNA in microtiter
plate
• DNA usually PCR
amplified
• Oligonucleotides can
also be spotted
DNA spotting II
• Pins dip into DNA solution in microtiter wells
• Robot moves pins with DNA to slides
• Robot “prints” DNA onto slide
– DNA sticks to slide by hydrostatic interactions
• Same spots usually printed at different locations
– Serves as internal control
• Pins washed between printing rounds
• Hundreds of slides can be printed in a day
Commercial DNA spotter
How microarrays are made:
Affymetrix GeneChips
• Oligonucleotides synthesized on silicon chip
– One base at a time
• Uses process of photolithography
– Developed for printing computer circuits
Affymetrix GeneChips
• Oligonucleotides
– Usually 20–25 bases in length
– 10–20 different oligonucleotides for each gene
• Oligonucleotides for each gene selected by
computer program to be the following:
– Unique in genome
– Nonoverlapping
• Composition based on design rules
• Empirically derived
Photolithography
• Light-activated chemical
reaction
– For addition of bases to
growing oligonucleotide
• Custom masks
– Prevent light from reaching
spots where bases not
wanted
• Mirrors also used
– NimbleGen™ uses this
approach
lamp
mask
chip
Example: building oligonucleotides
by photolithography
light
• Want to add nucleotide G
• Mask all other spots on
chip
• Light shines only where
addition of G is desired
• G added and reacts
• Now G is on subset of
oligonucleotides
Example: adding a second base
• Want to add T
• New mask covers spots
where T not wanted
• Light shines on mask
• T added
• Continue for all four bases
• Need 80 masks for total
20-mer oligonucleotide
light
Ink-jet printer microarrays
– Ink-jet printhead draws up DNA
– Printhead moves to specific location on solid
support
– DNA ejected through small hole
– Used to spot DNA or synthesize oligonucleotides
directly on glass slide
– Use pioneered by Agilent Technologies, Inc.
Comparisons of microarrays
Comparison of microarray
hybridization
• Spotted microarrays
– Competitive hybridization
• Two labeled cDNAs hybridized to same slide
• Affymetrix GeneChips
– One labeled RNA population per chip
– Comparison made between hybridization
intensities of same oligonucleotides on
different chips
Target labeling: fluorescent
cDNA
• cDNA made using
reverse transcriptase
• Fluorescently labeled
nucleotides added
• Labeled nucleotides
incorporated into
cDNA
Target labeling: cRNA + biotin
• cDNA made with
reverse
transcriptase
• Linker added with
T7 RNA polymerase
recognition site
• T7 polymerase
added and biotin
labeled RNA bases
• Biotin label
incorporated into
cRNA
+
Labels
• Cy3 and Cy5
– Fluoresce at different wavelengths
– Used for competitive hybridization
• Biotin
– Binds to fluorescently labeled avidin
– Used with Affymetrix GeneChips
Spotted-microarray
hybridization
• Control and experimental cDNA labeled
– One sample labeled with Cy3
– Other sample labeled with Cy5
• Both samples hybridized together to
microarray
• Relative intensity determined using
confocal laser scanner
Scanning of microarrays
laser
• Confocal laser
scanning microscopy
• Laser beam excites
each spot of DNA
• Amount of
fluorescence detected
• Different lasers used
for different
wavelengths
– Cy3
– Cy5
detection
Analysis of hybridization
• Results given as
ratios
• Images use colors:
Cy3 = Green
Cy5 = red
Yellow
– Yellow is equal
intensity or no change
in expression
Example of spotted microarray
• RNA from irradiated
cells (red)
• Compare with
untreated cells (green)
• Most genes have little
change (yellow)
• Gene CDKN1A: red =
increase in expression
• Gene Myc: green =
decrease in expression
CDKNIA
MYC
-Flash animation
-YouTube video
Analysis of cell-cycle regulation
• Yeast cells stopped at
different stages of cell
cycle
– G1, S, G2, and M
• RNA extracted from
each stage
• Control RNA from
unsynchronized
culture
Results of yeast cell-cycle analysis
• 800 genes identified whose expression
changes during cell cycle
• Grouped by peak expression
• M/G1, G1, S, G2, and M
• Four different treatments used to
synchronize cells
– All gave similar results
• Results from Spellman et al., 1998; Cho
et al., 1998
Cell-cycle regulated genes
Alpha
• Each gene is a line on
the longitudinal axis
• Treatments in
different panels
• Cell-cycle stages are
color coded at top
• Vertical axis groups
genes by stage in
which expression
peaks
cdc15
cdc28
Elu
M/G1
G1
S
G2
M
Brown and Botstein, 1999
Affymetrix GeneChip
experiment
• RNA from different types of brain tumors
extracted
• Extracted RNA hybridized to GeneChips
containing approximately 6,800 human
genes
• Identified gene expression profiles specific
to each type of tumor
Profiling tumors
• Image portrays gene
expression profiles
showing differences
between different
tumors
• Tumors:
MD (medulloblastoma)
Mglio (malignant glioma)
Rhab (rhabdoid)
PNET (primitive
neuroectodermal tumor)
• Ncer: normal cerebella
• Gene expression
differences for
medulloblastoma
correlated with response
to chemotherapy
• Those who failed to
respond had a different
profile from survivors
• Can use this approach to
determine treatment
60 different samples
Cancer diagnosis by microarray
Analysis of microarray results
• Inherent variability: need for repetition
– Biological and technical replicates
• Analysis algorithms
– Based on statistical models
• Means of generating hypotheses that need
to be tested