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Methods used to studygene expression
Slot blotsNorthern blots
In situ hybridizationRNA protection
assayPrimer extension
dd-RTPCRRT-PCR
Quantatitative RT-PCR (real-time)
Microarrays
Northern Blotting
Detection of specific RNA molecules
•Isolate total RNA•Purify poly A+ RNA if necessary
•Separate RNA fragments by Agarose Gel Electrophoresis
•Visualise
•Blot onto Membrane
Probe for specific fragments (RNA molecule)
• Label probe
• Hybridise to membrane
• Carry out washing at desired stringency
• Detect using suitable system
Ribosomal RNA
tRNA
Western and Northern analysis of alternative oxidase expression
Western
Northern
Northern
AOX3
AOX2
AOX3
AOX2
5 7 10 14 20Cotyledon Age
(days)
Probe - Gene of Interest
If known fine but what if do not have sequence
Use heterelogous Probe ?
Clone Gene of interest - variety of means
Label Probe Variety of methods
• Nick Translation• Random primer labelling with Klenow• PCR labelling
•Label with What ?
•Nucelotide that has a tag•Chromogenic•Radioactive•Luminescence•Fluorescence
Detection
All comes down to sensitivity
How much of the molecule will be present and is you labelling and detection system sensitive enough to detect
•Sensitivity of Northern blotting is an issue•Amount of RNA required is large•How comparative are different blots - can different blots on same gene be compared and can gene be compared
These issues have led to the development of other approaches to measure gene expression
AAAAAAA5’mRNAReverse TranscriptaseOligo dTDNA5’TTTTTTTTaq PolymeraseGene specific primer (1)TTTTTTT5’3’Taq PolymeraseGene specific primer (2)3’3’5’30 X
Reverse Transcriptase Polymerase Chain Reaction(RT-PCR)
Reverse TranscriptaseScheme
RT-PCR and biological relevance
RT-PCR (micro arrays, northern blots) measure steady state mRNA levels
RT-PCR (micro arrays, northern blots) do NOT measure transcription, mRNA stability or gene expression levels
fragment ofchromosomal DNA
DNA amplification:Polymerase Chain Reaction (PCR)
heat to separate,add primers
+ DNA polymerasedATP, dCTP, dGTP, dTTP
DNA synthesis from primersCycle 1
after 15 cycles, 32,768 copies; after 30 cycles, 1,073,741,824 copies
etc,
Cycle 2heat to separate,add primers
DNA synthesis from primers
etc,
etc,
etc,
Exponential increase is limited
Linear increase follows exponential
Eventually plateaus
Cycle #
Theoretical
Real Life
Log Target
DNA
Reality Check
The starting material for gene expression studies is RNA. For real time RT-PCR EVEN quality of RNA required. The RNA can either be:
Total RNA mRNA (poly A+)
rRNA: Building of ribosomes: machinery for synthesizing proteins by translating mRNA. Main
constituent of total RNA. 4 kinds, in eukaryotes, these are 18S rRNA, 28S, 5.8S, and 5S rRNA
mRNA: Translated into a polypeptide. mRNA can be purified using oligo d-T primers attached to a resin (polyA purified RNA)
tRNA: RNA molecules that carry amino acids to the growing polypeptide.
snRNA: The primary transcripts for mRNA, rRNA, and tRNA produces large precursor molecules, must be processed within the nucleus to produce the functional molecules for export to the cytosol. Some of these processing steps are mediated by snRNAs.
snoRNA: RNAs that help process ribosomal RNA (rRNA) molecules.
miRNA: These are tiny (~22 nts) RNA molecules that appear to regulate the expression of mRNA molecules.
What is Total RNA?
-All available RNA in the cell.
The RNA is transcribed to cDNA using reverse transcriptase and oligo dT primers, random hexamers or gene specific primers. This cDNA is then used in a real-time PCR reaction to determine the initial amount of RNA put in the RT (reverse transcriptase!) reaction.
What is the large assumption in making the above calculations?
The assumption:
All of the RT reactions occur at the same efficiency for all samples.
Any problems with this?
AAAAAAAAAAA
TTTTTTTTTTTT
TTTTTTTTTTTT
TTTTTTTTTTTT
F
R
F
Oligo dT primer
R
Reverse transcription
cDNA formation
cDNA
mRNA
PCR amplification
PCR product
RT-PCR
Reverse transcriptases
MMLV (Moloney Murine Reverse Transcriptase):
Lower activity temp; 37 CLower intrinsic Rnase H activity
AMV (Avian Myoblastosis Virus):
Higher activity temp 41 CHigher intrinsic Rnase H activity
Tth (therus thermophilus):
Both RT and DNA polymeraseHigh activity temp, 68-74 CSignificant less efficient than either above
Quantifying cDNA accurately is difficult - lets you know how efficient RT was carried out
- Direct - Spike with 32P-labeled dNTP (dCTP) precipitate and calculate mass of cDNA synthezised
- Indirect - Determine Ct level for a specific sample and gene of choice and use that as a standard for further synthesis steps = Housekeeping gene (Normalise)
Pure RNA samples,genomic DNA contamination and pseudo genes
•Treat samples when possible using RNase free DNase
•Design primers to bind to different exons or if possible over exon/exon junctions
The PCR Reaction
5’
5’
3’
3’
d.NTPs
Thermal Stable DNA Polymerase
Primers5’
3’5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Add Master Mix and Sample
Denaturation
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Annealing
Add to Reaction Tube
Extension
5’ 3’
5’3’5’ 3’
5’3’
Extension Continued
5’ 3’
5’3’5’
5’
Taq
Taq
3’
5’3’
Taq
Taq5’
5’
Repeat
The PCR Reaction
THE PCR REACTION
5’3’5’ 3’
5’3’5’
3’
5’5’3’
3’
5’3’5’ 3’
Cycle 2
4 Copies
Cycle 3
8 Copies
5’3’5’
3’
5’3’5’ 3’
5’3’5’ 3’
5’3’5’ 3’
5’3’5’ 3’
5’3’3’5’
5’3’5’ 3’
5’3’5’ 3’
PCR - Powerful Tool!! PCR technology is an essential tool for Molecular Biology
PCR allows rapid and reproducible amplification of a specific sequence of DNA
PCR technology is responsible for accelerating Genetic Discoveries
HOWEVER, IT COULD DO MORE!
Exponential increase is limited
Linear increase follows exponential
Eventually plateaus
Cycle #
Theoretical
Real Life
Log Target
DNA
Reality Check
PCR - Powerful Tool!! PCR technology is an essential tool for Molecular Biology
PCR allows rapid and reproducible amplification of a specific sequence of DNA
PCR technology is responsible for accelerating Genetic Discoveries
HOWEVER, IT COULD DO MORE!
R
elative
Relative
Fluorescence
Fluorescence
End Point End Point MeasurementsMeasurements
In Addition
In Addition
96 Replicates of 96 Replicates of identicalidentical reactions reactions have very different have very different individual efficiencies individual efficiencies by the end of the by the end of the reactionreaction
Threshold Cycle, Ct, of the same 96 replicates shows nearly identical values
Real Time PCR incorporates the ability to directly measure and quantify the
reaction while amplification is taking
place.
What is Real Time PCR?
0
0.4
0.8
1.2
1.6
2
0 10 20 21 30 40
Threshold
Baseline
Sample
CCTT
What is Threshold Cycle (CT)?
Threshold Cycle, Ct, is a reliable
indicator of initial copy number
What Detection Strategies are
available?
Dyes Intercalating Dyes are inexpensive compared to hybridization probes.
A dye based strategy allows one to take a “big picture” - that is - get a general confirmation of amplification.
Russ Higuchi demonstrated the key principle of Real Time PCR using Ethidium Bromide - EtBr fluoresces 25 times more brightly EtBr fluoresces 25 times more brightly when bound to dsDNA when bound to dsDNA
SYBR Green, a more sensitive dye is an even more attractive approach SYBR Green fluoresces 200 times more SYBR Green fluoresces 200 times more brightly when bound to dsDNAbrightly when bound to dsDNA
Sybr Green binding
It can intercalate into DNA - but this does not result in increase in Flouresence
Binding to minor groove of ds DNA results in Increase in Fluorescence
Can bind ssDNA - no increase in binding
Fluorescence can be quenched by impurities in RNA sample
Binding influenced by:i) Salt concentration (NaCl, MgCl2)
ii)Concentration of ds DNA
5’
5’
3’
3’
d.NTPs
Thermal Stable DNA Polymerase
Primers5’
3’5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Add Master Mix and Sample
Denaturation
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Annealing
Reaction Tube
Dyes
Intercalation Dyes
Taq ID
Extension
5’ 3’
5’3’5’ 3’
5’3’
Extension ContinuedApply Excitation
Wavelength
5’ 3’
5’3’5’
5’
Taq
Taq
3’
5’3’
Taq
Taq5’
5’
Repeat
Dyes
ID ID
ID IDID
ID ID ID
ID ID
Hybridization Probes
Cleavage Based Assay - TaqMan Assays
Displaceable Probe Assays Molecular Beacons Dual oligo FRET probes
Probes incorporated directly into the primers Amplifluor Scorpions
Today Hybridization Probe Strategies fall into three main categories:
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Identify promoter motifs that distinguish gene clusters
TaqManTM
5’
5’
3’
3’
d.NTPs
Thermal Stable DNA Polymerase
Primers5’
3’5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Add Master Mix and Sample
Denaturation
5’
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5’
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5’
3’5’
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Annealing
Reaction Tube
Taq
5’ 3’
R Q
Probe
5’ 3’
R Q
5’ 3’
5’3’
TaqManTM
5’3’
RQ
Extension Step
5’ 3’
1. Strand DisplacementTaq3’
Q
R
5’
5’ 3’3’
Q Taq
R
5’ 2. Cleavage
3. PolymerizationComplete5’ 3’
QTaqR
3’ 5’
4. Detection5’ 3’
3’
QTaqR
5’
R
5’
5’
3’
3’
d.NTPs
Thermal Stable DNA Polymerase
Primers5’
3’5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Add Master Mix and Sample
Annealing
Reaction Tube
Molecular Beacons
Denaturation
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Taq
5’ 3’
R Q
R Q
MolecularBeacon
5’ 3’
5’3’
Extension Step5’ 3’
1. Strand DisplacementTaq5’
2. PolymerizationComplete
Probe Silent
Molecular Beacons
5’3’
RQ
Detection
5’3’
RQ
5’ 3’
3’ 5’
TaqR Q
MolecularBeacon
FRET Probes
5’ 3’
5’3’
3’
D R
5’ Detection
Extension Step5’ 3’
1. Strand DisplacementSystem Silent
2. PolymerizationComplete
System Silent
5’ 3’
3’ 5’
Taq
3’
D
R1-5 bases
D
R
Taq5’
R
5’
Primer Based
3’5’
R Q
Heat
Incorporation
R Q
Primer Based
5’
Extension 23’5’
R
Q
5’ 3’ 5’R Q Annealing/Extension 13’
QR
3’
5’ 3’
5’
Detection
