Transcript Lecture 7 • Assignment 2 • RT-PCR process and principles • Etiolation

Lecture 7
• Assignment 2
• RT-PCR process and principles
• Etiolation
• DET1
• Discussion of Article 4
Assignment 2
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Sample 1 General Hans Canis
Sample 2 Field Marshall Heinrich Rattus
Sample 3 Mrs. Heidi Rattus
Sample 4 Miss. Gertrude Musca
Sample 5 Mr. Ivan Apis
Sample 6 Blood on the envelope
Sample 7 Hair found in Gertrude’s apartment.
Assignment 2
Assignment 2 due Nov 10, 2008
Experimental set up
1. wild-type grown in the light
2. wild-type grown in the dark
3. det1 grown in the light
4. det1 grown in the dark
-Extract RNA
-RT mRNA
-PCR cDNA
Picture of Plants (WT and mutant)
What are the expression levels of
light regulated genes (chlorophyll
a/b binding proteins CHAB and CLAB)
using tubulin as loading control.
CHAB
CLAB
CHAB or
CLAB
TUB
Trizol Method
• Why do we grind in liquid N2?
• What does the Guanidium Thiocyanate
do?
• What does the Phenol do?
• What does the Chloroform do?
• What does the isopropanol do?
Isolation of RNA using Commercial Trizol Reagent
Trizol:-mono-phasic solution of phenol and guanidine isothiocyanate
-lyses cells and dissolves cell components while maintaining integrity of RNA
Liquid N2
Trizol
Homogenize
Phenol
Aq Isopropyl
alcohol
Chloroform
Org Ppt RNA
75%
EtOH
DEPC
H2O RNA
Chloroform: separate aqueous and organic phases (RNA is in the top aqueous phase)
Isopropanol: Precipitates RNA
75% Ethanol: Wash RNA pellet
DEPC (Diethylpyrocarbonate) water: Resuspend RNA pellet in RNAse-free water
DEPC interacts with the active site histidine residue of RNAse and irreversibly
inactivates RNAses.
RNA preparation:
Considerations
• Yield: What controls yield?
• RNases: What do we need to control
this?
• Quality of mRNA: What do we mean?
• DNA contamination: Why is this
important in RT-PCR?
Dangers this week
• TRIzol is very dangerous and can cause
severe chemical burns.
• Liquid N2 is very cold and can burn the
skin; be careful and no fooling around.
Original Northern blot
Pepper et al., 1994 Cell 78, 109-116
RT-PCR: reverse transcriptase
COMPONENTS TO ADD FOR REVERSE TRANSCRIPTION
component
1
2
3
4
wild-type total
RNA; light
5ml
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det1 total RNA;
light
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5ml
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wild-type total
RNA ; dark
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5ml
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det1 total RNA;
dark
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5ml
water
5ml
5ml
5ml
5ml
oligo dT primer
2ml
2ml
2ml
2ml
Total volume
12ml
12ml
12ml
12ml
COMPONENTS TO ADD FOR REVERSE TRANSCRIPTION
Component
Volume
5X Superscript
buffer
4ml
0.1 DTT
2ml
10mM dNTP
1ml
Final volume
20ml
Add 0.5 μl Superscript reverse transcriptase.
What do you think is in the RT
buffer?
Multiplex PCR
• What is multiplex PCR?
• What are the top two considerations
when performing a multiplex PCR?
What is in our multiplex PCR?
• What is the control and what are light
regulated?
What is Multiplex PCR? (Combinatorial PCR)
Amplifying various genes simultaneously (One template with multiple primer pairs)
CHAB
Multiplex PCR is used for:
1.
2.
3.
4.
5.
DNA polymorphisms
Mutations
Detection of Pathogens
Genetically modified organisms
Quantitative RT-PCR
CLAB
CHAB or
CLAB
TUB
Why are we amplifying (CHAB or CLAB) and tubulin simultaneously?
RT-PCR
• What is the problem with having DNA
contamination?
• Why is a set of exon primers that span
an intron a good idea?
Hint!!!
What will be the outcome of an
RT-PCR experiment if there is
DNA contamination?
Nice figure taken from Ann. Rev. Cell Dev. Biol
13, 203
Etiolated stage
• Long hypocotyl (primary stem)
• Apical hook
• Unopened cotyledons (embryonic leaves)
De-etiolated
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Hypocotyl growth decreases
Apical hook opens
Cotyledons expand
Chloroplasts develop
A new gene expression program is
induced
Comparison of dark-grown (etiolated) and light-grown (de-etiolated) seedlings
Etiolated characteristics
De-etiolated characteristics
No leaf growth
Leaf growth promoted
No chlorophyll
Chlorophyll produced
Rapid stem elongation
Stem elongation suppressed
Limited radial expansion of stem
Radial expansion of stem
Limited root elongation
Root elongation promoted
Limited production of lateral roots
Lateral root development accelerated
New Gene Expression Program is induced
• Etiolation and de-etiolation are normal
stages of plant development.
• After germination there may be a lack of
light and the seedling grows to find the
light. Etiolated.
• When sufficient light is found the
seedling de-etiolates a developmental
process that optimizes the body plan for
photosynthesis.
Picture of DET1 function
DET1 is a nuclear protein with a nuclear localization sequence.
DET1 is part of a protein complex with COP10 and DDB1 called CDD
CDD stimulates ubiquitin-conjugating enzymes.
Ubiquitination of HY5 (required for normal light development)
-targets it for degradation.
DET1 function in light and dark
Why have det1 mutants lost
this response?
No DET1; HY5 is not degraded
and can not initiate etiolated
growth.
Det1 in Humans?
• Human de-etiolated-1 regulates c-Jun
by assembling a CUL4A ubiquitin ligase.
Science 303, 1371-1374 (2004).
• Jun is an oncogene!
Discussion of Article 4