Transcript A501, CCQuirk
Slide 1
Measuring Gene Expression
Chris Quirk, PhD
Medical Sciences Program
JH206, 856-2808
A501, CCQuirk
Slide 2
Innovations in Technology
● Industrial revolution
● 1750 – 1830
● Originated in Great Britain
● Transformation from a largely rural population making a living
almost entirely from agriculture to a town-centered society
engaged increasingly in factory manufacture
A501, CCQuirk
Slide 3
Innovations in Technology
●
Computer revolution (started during World War II)
● Colossus I, a computer designed by the British to break Nazi military
codes
● Mark I, first fully automated ‘computer’ built by IBM for Harvard
(calculator)
● could store 72 numbers, each 23 decimal digits long. It could do
three additions or subtractions in a second
● a multiplication took 6 seconds, a division took 15.3 seconds, and a
logarithm or a trigonometric
function took over one minute
A501, CCQuirk
Slide 4
Innovations in Technology
●
Genomics revolution
● term “genome” was created by merging the words genes and
chromosomes in 1920
● refers to all the DNA in an organism, including its genes
● term “genomics” was coined in 1986 to describe
the scientific discipline of mapping, sequencing,
and analyzing genomes
● name-sake to a scientific journal that was
initiated at that time, Genomics
● the goal of genomics is to make biological and
functional sense of raw genetic information
● In 2000, Francis Collins (NIH) and J. Craig Venter
(Celera Genomics) announce a working draft of
the human genome
A501, CCQuirk
Slide 5
Human Genome Project
● International effort began in October 1990
● Project was planned to last 15 years, but rapid technological
●
●
advances accelerated the completion to 2003
Project goals
● Determine the complete sequence of the 3 billion DNA bases in
the human genome
● Identify all human genes
● Make the genes accessible for further biological study
Genomes from other species also sequenced
● Mice
● Fruit flies
● Zebra fish
● Yeast
● Nematodes
● Plants
● Many microbial organisms and parasites
A501, CCQuirk
Slide 6
Central Dogma of Molecular Biology
Transfer of genetic material:
1. Structural changes in
chromatin are activated
2. Initiation of gene
transcription
3. Processing of emergent
RNA transcript
4. Transport of mature mRNA
to cytoplasm
5. Translation of the encoded
polypeptide
A501, CCQuirk
Slide 7
Basics of DNA
●
●
●
DNA is a long chain of molecules that takes on a double helix shape
● adenine (A), guanine (G), thymine (T) and cytosine (C)
DNA is the genetic blue print for all cellular processes
Whereas DNA is the script for making RNA and proteins, RNA directs the
production of all proteins
A501, CCQuirk
Slide 8
Transcription
A501, CCQuirk
Slide 9
Breeding sheep: Short day vs. long day
● Length of the estrous cycle 13 to 19 days, average 17 days
● Phases of the estrous cycle are proestrus, estrus, metestrus,
●
and diestrus
● Estrus is the period of time when the ewe is receptive to the ram
and will stand for mating (lasts approximately 24 to 36 hours)
Estrous cycles are usually affected by the seasons
● The number of hours daily that light enters the eye of the animal
affects the brain, which governs the release of certain precursors
and hormones
● Most sheep are seasonally polyestrus and short-day breeders
● Begin to exhibit estrus when length of day begins decreasing
● Most natural time for sheep to breed in the U.S. and Canada
is the fall (Oct-Nov)
A501, CCQuirk
Slide 10
What makes sheep short-day breeders?
14h light : 10h dark
10h light : 14h dark
anestrus
estrous cycling
cells in hypothalamus
A501, CCQuirk
Slide 11
Why does expression of p8 increase
tumorigenic potential?
p8-KD-LβT2
1.0
% tumor free
C-LβT2
(express p8)
0.8
0.6
C-LβT2 (n=13)
p8-KD-LβT2 (n=22)
censored
0.4
0.2
0.0
0
50
100 150 200 250 300
days post-injection
A501, CCQuirk
Slide 12
●
●
A501, CCQuirk
LßT2
C-LßT2
p8-OE-LßT2
●
method for detection and
quantification of mRNA levels
provides a direct relative comparison
of message abundance between
samples on a single membrane
preferred method for determining
transcript size and for detecting
alternatively spliced transcripts
Limitations:
● if RNA samples are even slightly
degraded, the quality of the data
and the ability to quantitate
expression are severely
compromised
● less sensitive than nuclease
protection assays and RT-PCR
● difficulty associated with multiple
probe analysis
T3-1
● Developed in 1977 as standard
p8-KD-LßT2
Northern Blot Analysis
Kb
2.37
ßactin
18S
1.35
p8
CMV-p8
0.24
–
– ++
+
+ p8 mRNA
~30,000 genes!
Slide 13
Gene Expression Profiling
● DNA Chips, Genome Chips, Bio chip, and cDNA arrays are all
similar terms for one the most incredible and influential technologies
in the area of genetic research known today – the GeneChip
microarray
● Affymetrix arrays are built using the
same type of technology that is used
to manufacture semiconductor chips,
but rather than etching miniature
circuits, Affymetrix builds millions of
strands of DNA.
A501, CCQuirk
Slide 14
When a single strand of DNA
(ATCATG) matches a strand of RNA
(UAGUAC), the two strands are
"complementary" and will stick to
each other. However, if the bases
aren't complementary, they won't fit
together.
Affymetrix microarrays use base pairing attraction (hybridization) to help
researchers identify what RNA sequences are present in a sample, and
this then tells them how strongly those genes are being expressed.
A501, CCQuirk
Slide 15
Making a GeneChip…
●
●
●
Build a short DNA strand — a probe — on the surface of a glass chip
Sequence of the probe is compared to the rest of the human genome
to make sure it doesn't match anywhere else
For every probe, a mismatch probe is also produced
A501, CCQuirk
Slide 16
● In all, there are 22 different probes, or data points, used to make
●
sure that the microarray is detecting the correct piece of RNA
By measuring that RNA with 11 probe pairs we can be
absolutely certain that the gene we think is expressed actually is
A501, CCQuirk
Slide 17
Features
●
●
●
●
The surface of the Affymetrix array is like a giant
checkerboard that has been shrunk down to the
size of a thumbnail. Each square (feature) on the
checkerboard holds millions of copies of one
unique type of probe
The most recent Affymetrix human genome array
has more than 1.3 million squares/features
● Represents about 47,000 different RNAs
Each feature on the array is about 11 microns
across (~one-fifth the width of a human hair)
Affymetrix builds these probes one molecule at a
time, using the same type of manufacturing
technology that is used to build computer
semiconductors
● The molecules are built one layer (base) at a
time, one stacked on top of another, like
checkers
A501, CCQuirk
Slide 18
Getting the RNA Ready
● Extract RNA from the sample
● Amplify it
● Copying the RNA allows it to be more easily detected on the
●
●
array
At the same time the RNA is copied, molecules of biotin (orange
cups) are attached to each strand
Fragment the RNA
A501, CCQuirk
Slide 19
A501, CCQuirk
Slide 20
A501, CCQuirk
Slide 21
Genome-wide arrays
long day
short day
common
genes
genes unique
to long day
A501, CCQuirk
genes unique
to short day
Slide 22
Genome-wide arrays
long day
short day
Number of Unique Genes
600
500
400
300
200
100
0
A501, CCQuirk
1
GeneChip Number
Slide 23
Genome-wide arrays
long day
short day
Number of Unique Genes
600
500
400
300
200
100
0
A501, CCQuirk
1
2
GeneChip Number
Slide 24
Genome-wide arrays
long day
short day
Number of Unique Genes
600
500
400
300
200
100
0
A501, CCQuirk
1
2
3
GeneChip Number
Slide 25
Genome-wide arrays
long day
short day
Number of Unique Genes
600
500
400
300
200
100
0
A501, CCQuirk
1
2
3
4
GeneChip Number
Slide 26
Comparing Expression
short day (cycling)
long day (anestrus)
1
2
3
4
5
● Researchers construct “heat maps”; graphical displays that color code
gene expression
● Increased expression is color coded in red
● Decreased expression in blue
A501, CCQuirk
Slide 27
RNA Extraction Laboratory
Organized by Christina Million Passe
Kate Brannon
Crystal White
Laurel Bender
JH209 & 211
A501, CCQuirk
Slide 28
RNA Isolation Protocol
A501 - Techniques in Reproductive Diversity
28 November 2006
A501, CCQuirk
Slide 29
Step 1: Clean work area
Notes:
• RNA is very susceptible to degradation by ribonucleases
(RNases)
• RNases can be found almost everywhere in our environment, so
we need to clean everything before we harvest RNA
• RNase Away - noncarcinogenic RNase, DNase, and DNA
decontaminant (proprietary formulation)
Protocol:
• Spray work area with 70% ethanol, then RNase Away, then
ddH2O
A501, CCQuirk
Slide 30
Step 2: Homogenize tissue in TRIzol
Notes:
• TRIzol - mixture of phenol and guanidine isothiocyanate (GTC)
• Phenol - organic solvent that lyses cells and denatures
proteins
• GTC - inactivates RNases
Protocol:
• Add TRIzol to tissue sample and homogenize with a tissue
homogenizer tip
– Work quickly! Tissues will begin to release RNases as soon
as you begin homogenizing!
• Add additional TRIzol (total volume = 500 L) and incubate 5
minutes
A501, CCQuirk
Slide 31
Step 3: Separate phases with chloroform
Notes:
• Chloroform - organic solvent that further denatures proteins and
allows aqueous and organic phases to separate
– Proteins stay in organic phase (bottom)
– RNA goes to aqueous phase (top)
– DNA goes to either, depending on pH
• Low pH (like TRIzol), DNA stays in organic phase
• High pH, DNA goes to aqueous phase
• Phase Lock GelTM (PLG) - migrates between aqueous and
organic phases upon centrifugation
– Aqueous phase can be easily decanted
A501, CCQuirk
Slide 32
Step 3: Separate phases (continued)
Protocol:
• Add chloroform to tissue homogenate, mix vigorousy, and
incubate 3 minutes
• During incubation, pellet gel in a PLG tube
• Separate phases by centrifugation
A501, CCQuirk
Slide 33
Step 4: Precipitate RNA
Notes:
• Nucleic acids are insoluble in concentrated alcohols
Protocol:
• Decant aqueous phase to a clean tube
• Add 100% isopropanol, mix, and incubate 10 minutes
• Pellet RNA by centrifugation
A501, CCQuirk
Slide 34
Step 5: Wash RNA
Notes:
• Washing with less concentrated alcohols will remove impurities
(residual salts, etc.) from RNA pellet
• Alcohols must be diluted in RNase-free H2O
– Diethyl pyrocarbonate (DEPC) is a histidine-specific
alkylating agent that destroys enzymatic activity
– Treating H2O with 0.1% DEPC inactivates RNases
Protocol:
• Remove supernatant
• Wash pellet with ice cold 75% ethanol diluted in DEPC H2O
• Pellet RNA by centrifugation
A501, CCQuirk
Slide 35
Step 6: Redissolve RNA
Notes:
• Before redissolving, RNA must be dried to remove residual
ethanol, which will make the pellet insoluble
• Over-drying the pellet will make it difficult to dissolve
• To avoid over-drying, we dry RNA until a “halo” (clear, dry circle)
forms around the outside of the pellet, but the center remains
white
Protocol:
• Remove as much supernatant as possible
• Dry RNA until a “halo” forms
• Redissolve in DEPC H2O and place on ice
A501, CCQuirk
Slide 36
Steps 7-8: Determine RNA concentration &
verify integrity
Notes:
• Intact RNA will yield two predominant bands when separated by
electrophoresis, the 28S and 18S ribosomal subunits
Protocol:
• Determine RNA concentration according to the instructions of
your group leader
• Run 1 g RNA per lane on a 1% agarose + ethidium bromide
gel and separate RNA by electrophoresis
• Take a picture of your gel using the camera/UV box
A501, CCQuirk
Slide 37
Things to do while you’re waiting for your gel to
run…
• Join Crystal as she performs mouse necropsies to collect
organs of reproductive relevance
– This option is completely voluntary!
• Learn from Kate how we obtain our GeneChip data from the
microarray data portal and see the types of things you can do
with the data on the portal’s website
• Sort through some of the Quirk Lab’s GeneChip data and see if
you can find your favorite gene(s)
• Read the “Red Book” protocol for RNA isolation
– Current Protocols, Volume 1, Section 4.2.1
• Look over the TRIzol and PLG protocols
– Find out how you can use these to obtain protein and DNA
A501, CCQuirk
Measuring Gene Expression
Chris Quirk, PhD
Medical Sciences Program
JH206, 856-2808
A501, CCQuirk
Slide 2
Innovations in Technology
● Industrial revolution
● 1750 – 1830
● Originated in Great Britain
● Transformation from a largely rural population making a living
almost entirely from agriculture to a town-centered society
engaged increasingly in factory manufacture
A501, CCQuirk
Slide 3
Innovations in Technology
●
Computer revolution (started during World War II)
● Colossus I, a computer designed by the British to break Nazi military
codes
● Mark I, first fully automated ‘computer’ built by IBM for Harvard
(calculator)
● could store 72 numbers, each 23 decimal digits long. It could do
three additions or subtractions in a second
● a multiplication took 6 seconds, a division took 15.3 seconds, and a
logarithm or a trigonometric
function took over one minute
A501, CCQuirk
Slide 4
Innovations in Technology
●
Genomics revolution
● term “genome” was created by merging the words genes and
chromosomes in 1920
● refers to all the DNA in an organism, including its genes
● term “genomics” was coined in 1986 to describe
the scientific discipline of mapping, sequencing,
and analyzing genomes
● name-sake to a scientific journal that was
initiated at that time, Genomics
● the goal of genomics is to make biological and
functional sense of raw genetic information
● In 2000, Francis Collins (NIH) and J. Craig Venter
(Celera Genomics) announce a working draft of
the human genome
A501, CCQuirk
Slide 5
Human Genome Project
● International effort began in October 1990
● Project was planned to last 15 years, but rapid technological
●
●
advances accelerated the completion to 2003
Project goals
● Determine the complete sequence of the 3 billion DNA bases in
the human genome
● Identify all human genes
● Make the genes accessible for further biological study
Genomes from other species also sequenced
● Mice
● Fruit flies
● Zebra fish
● Yeast
● Nematodes
● Plants
● Many microbial organisms and parasites
A501, CCQuirk
Slide 6
Central Dogma of Molecular Biology
Transfer of genetic material:
1. Structural changes in
chromatin are activated
2. Initiation of gene
transcription
3. Processing of emergent
RNA transcript
4. Transport of mature mRNA
to cytoplasm
5. Translation of the encoded
polypeptide
A501, CCQuirk
Slide 7
Basics of DNA
●
●
●
DNA is a long chain of molecules that takes on a double helix shape
● adenine (A), guanine (G), thymine (T) and cytosine (C)
DNA is the genetic blue print for all cellular processes
Whereas DNA is the script for making RNA and proteins, RNA directs the
production of all proteins
A501, CCQuirk
Slide 8
Transcription
A501, CCQuirk
Slide 9
Breeding sheep: Short day vs. long day
● Length of the estrous cycle 13 to 19 days, average 17 days
● Phases of the estrous cycle are proestrus, estrus, metestrus,
●
and diestrus
● Estrus is the period of time when the ewe is receptive to the ram
and will stand for mating (lasts approximately 24 to 36 hours)
Estrous cycles are usually affected by the seasons
● The number of hours daily that light enters the eye of the animal
affects the brain, which governs the release of certain precursors
and hormones
● Most sheep are seasonally polyestrus and short-day breeders
● Begin to exhibit estrus when length of day begins decreasing
● Most natural time for sheep to breed in the U.S. and Canada
is the fall (Oct-Nov)
A501, CCQuirk
Slide 10
What makes sheep short-day breeders?
14h light : 10h dark
10h light : 14h dark
anestrus
estrous cycling
cells in hypothalamus
A501, CCQuirk
Slide 11
Why does expression of p8 increase
tumorigenic potential?
p8-KD-LβT2
1.0
% tumor free
C-LβT2
(express p8)
0.8
0.6
C-LβT2 (n=13)
p8-KD-LβT2 (n=22)
censored
0.4
0.2
0.0
0
50
100 150 200 250 300
days post-injection
A501, CCQuirk
Slide 12
●
●
A501, CCQuirk
LßT2
C-LßT2
p8-OE-LßT2
●
method for detection and
quantification of mRNA levels
provides a direct relative comparison
of message abundance between
samples on a single membrane
preferred method for determining
transcript size and for detecting
alternatively spliced transcripts
Limitations:
● if RNA samples are even slightly
degraded, the quality of the data
and the ability to quantitate
expression are severely
compromised
● less sensitive than nuclease
protection assays and RT-PCR
● difficulty associated with multiple
probe analysis
T3-1
● Developed in 1977 as standard
p8-KD-LßT2
Northern Blot Analysis
Kb
2.37
ßactin
18S
1.35
p8
CMV-p8
0.24
–
– ++
+
+ p8 mRNA
~30,000 genes!
Slide 13
Gene Expression Profiling
● DNA Chips, Genome Chips, Bio chip, and cDNA arrays are all
similar terms for one the most incredible and influential technologies
in the area of genetic research known today – the GeneChip
microarray
● Affymetrix arrays are built using the
same type of technology that is used
to manufacture semiconductor chips,
but rather than etching miniature
circuits, Affymetrix builds millions of
strands of DNA.
A501, CCQuirk
Slide 14
When a single strand of DNA
(ATCATG) matches a strand of RNA
(UAGUAC), the two strands are
"complementary" and will stick to
each other. However, if the bases
aren't complementary, they won't fit
together.
Affymetrix microarrays use base pairing attraction (hybridization) to help
researchers identify what RNA sequences are present in a sample, and
this then tells them how strongly those genes are being expressed.
A501, CCQuirk
Slide 15
Making a GeneChip…
●
●
●
Build a short DNA strand — a probe — on the surface of a glass chip
Sequence of the probe is compared to the rest of the human genome
to make sure it doesn't match anywhere else
For every probe, a mismatch probe is also produced
A501, CCQuirk
Slide 16
● In all, there are 22 different probes, or data points, used to make
●
sure that the microarray is detecting the correct piece of RNA
By measuring that RNA with 11 probe pairs we can be
absolutely certain that the gene we think is expressed actually is
A501, CCQuirk
Slide 17
Features
●
●
●
●
The surface of the Affymetrix array is like a giant
checkerboard that has been shrunk down to the
size of a thumbnail. Each square (feature) on the
checkerboard holds millions of copies of one
unique type of probe
The most recent Affymetrix human genome array
has more than 1.3 million squares/features
● Represents about 47,000 different RNAs
Each feature on the array is about 11 microns
across (~one-fifth the width of a human hair)
Affymetrix builds these probes one molecule at a
time, using the same type of manufacturing
technology that is used to build computer
semiconductors
● The molecules are built one layer (base) at a
time, one stacked on top of another, like
checkers
A501, CCQuirk
Slide 18
Getting the RNA Ready
● Extract RNA from the sample
● Amplify it
● Copying the RNA allows it to be more easily detected on the
●
●
array
At the same time the RNA is copied, molecules of biotin (orange
cups) are attached to each strand
Fragment the RNA
A501, CCQuirk
Slide 19
A501, CCQuirk
Slide 20
A501, CCQuirk
Slide 21
Genome-wide arrays
long day
short day
common
genes
genes unique
to long day
A501, CCQuirk
genes unique
to short day
Slide 22
Genome-wide arrays
long day
short day
Number of Unique Genes
600
500
400
300
200
100
0
A501, CCQuirk
1
GeneChip Number
Slide 23
Genome-wide arrays
long day
short day
Number of Unique Genes
600
500
400
300
200
100
0
A501, CCQuirk
1
2
GeneChip Number
Slide 24
Genome-wide arrays
long day
short day
Number of Unique Genes
600
500
400
300
200
100
0
A501, CCQuirk
1
2
3
GeneChip Number
Slide 25
Genome-wide arrays
long day
short day
Number of Unique Genes
600
500
400
300
200
100
0
A501, CCQuirk
1
2
3
4
GeneChip Number
Slide 26
Comparing Expression
short day (cycling)
long day (anestrus)
1
2
3
4
5
● Researchers construct “heat maps”; graphical displays that color code
gene expression
● Increased expression is color coded in red
● Decreased expression in blue
A501, CCQuirk
Slide 27
RNA Extraction Laboratory
Organized by Christina Million Passe
Kate Brannon
Crystal White
Laurel Bender
JH209 & 211
A501, CCQuirk
Slide 28
RNA Isolation Protocol
A501 - Techniques in Reproductive Diversity
28 November 2006
A501, CCQuirk
Slide 29
Step 1: Clean work area
Notes:
• RNA is very susceptible to degradation by ribonucleases
(RNases)
• RNases can be found almost everywhere in our environment, so
we need to clean everything before we harvest RNA
• RNase Away - noncarcinogenic RNase, DNase, and DNA
decontaminant (proprietary formulation)
Protocol:
• Spray work area with 70% ethanol, then RNase Away, then
ddH2O
A501, CCQuirk
Slide 30
Step 2: Homogenize tissue in TRIzol
Notes:
• TRIzol - mixture of phenol and guanidine isothiocyanate (GTC)
• Phenol - organic solvent that lyses cells and denatures
proteins
• GTC - inactivates RNases
Protocol:
• Add TRIzol to tissue sample and homogenize with a tissue
homogenizer tip
– Work quickly! Tissues will begin to release RNases as soon
as you begin homogenizing!
• Add additional TRIzol (total volume = 500 L) and incubate 5
minutes
A501, CCQuirk
Slide 31
Step 3: Separate phases with chloroform
Notes:
• Chloroform - organic solvent that further denatures proteins and
allows aqueous and organic phases to separate
– Proteins stay in organic phase (bottom)
– RNA goes to aqueous phase (top)
– DNA goes to either, depending on pH
• Low pH (like TRIzol), DNA stays in organic phase
• High pH, DNA goes to aqueous phase
• Phase Lock GelTM (PLG) - migrates between aqueous and
organic phases upon centrifugation
– Aqueous phase can be easily decanted
A501, CCQuirk
Slide 32
Step 3: Separate phases (continued)
Protocol:
• Add chloroform to tissue homogenate, mix vigorousy, and
incubate 3 minutes
• During incubation, pellet gel in a PLG tube
• Separate phases by centrifugation
A501, CCQuirk
Slide 33
Step 4: Precipitate RNA
Notes:
• Nucleic acids are insoluble in concentrated alcohols
Protocol:
• Decant aqueous phase to a clean tube
• Add 100% isopropanol, mix, and incubate 10 minutes
• Pellet RNA by centrifugation
A501, CCQuirk
Slide 34
Step 5: Wash RNA
Notes:
• Washing with less concentrated alcohols will remove impurities
(residual salts, etc.) from RNA pellet
• Alcohols must be diluted in RNase-free H2O
– Diethyl pyrocarbonate (DEPC) is a histidine-specific
alkylating agent that destroys enzymatic activity
– Treating H2O with 0.1% DEPC inactivates RNases
Protocol:
• Remove supernatant
• Wash pellet with ice cold 75% ethanol diluted in DEPC H2O
• Pellet RNA by centrifugation
A501, CCQuirk
Slide 35
Step 6: Redissolve RNA
Notes:
• Before redissolving, RNA must be dried to remove residual
ethanol, which will make the pellet insoluble
• Over-drying the pellet will make it difficult to dissolve
• To avoid over-drying, we dry RNA until a “halo” (clear, dry circle)
forms around the outside of the pellet, but the center remains
white
Protocol:
• Remove as much supernatant as possible
• Dry RNA until a “halo” forms
• Redissolve in DEPC H2O and place on ice
A501, CCQuirk
Slide 36
Steps 7-8: Determine RNA concentration &
verify integrity
Notes:
• Intact RNA will yield two predominant bands when separated by
electrophoresis, the 28S and 18S ribosomal subunits
Protocol:
• Determine RNA concentration according to the instructions of
your group leader
• Run 1 g RNA per lane on a 1% agarose + ethidium bromide
gel and separate RNA by electrophoresis
• Take a picture of your gel using the camera/UV box
A501, CCQuirk
Slide 37
Things to do while you’re waiting for your gel to
run…
• Join Crystal as she performs mouse necropsies to collect
organs of reproductive relevance
– This option is completely voluntary!
• Learn from Kate how we obtain our GeneChip data from the
microarray data portal and see the types of things you can do
with the data on the portal’s website
• Sort through some of the Quirk Lab’s GeneChip data and see if
you can find your favorite gene(s)
• Read the “Red Book” protocol for RNA isolation
– Current Protocols, Volume 1, Section 4.2.1
• Look over the TRIzol and PLG protocols
– Find out how you can use these to obtain protein and DNA
A501, CCQuirk