Functional Genomics Research Stream Plate Assays

Plate Assays
Current Results
Functional Genomics
Research Stream
Research Meeting: June 22, 2011
Differential Expression Experiments
30C
37C
DNA Damage
Results
+24
hours
Order:
Saccharomyces bayanus
Saccharomyces cerevisiae
Saccharomyces mikatae
Saccharomyces paradoxus
Saccharomyces cerevisiae MET18∆ (+ control)
Saccharomyces bayanus
Saccharomyces cerevisiae
Saccharomyces mikatae
30C
37C
30C
37C
+48
hours
+48
hours
Saccharomyces paradoxus
30C
48 to 120
hours
30C
YPD
(normal)
YPD + 0.05% MMS
(DNA damaging agent)
Katherine Luethcke, 2011
Hyper-osmotic
Stress
Order:
Saccharomyces bayanus
Saccharomyces cerevisiae
Saccharomyces mikatae
Saccharomyces paradoxus
Plate Assays
What’s the point?
+16
hours
YPD
(normal)
YPD + 1M Sorbitol
Hilary Rabago, 2011
Stress
• Heat
______________
Damage with MMS
• DNA
Katie & Allison & ______________
Stress (23C)
• Cold
David
O
• HMichael
Z’s & ______________
2
2
• Hyper & Hypo Osmotic Stress
Wonder Twins & ______________
• Amino Acid Starvation
Elise & Neha
Post-Plate Assays
What next?
RT-PCR
RT-PCR
Gene X
Transcription
Gene X RNA
How does this help?
AAAAAAAAAAAAAA
RT
Gene X cDNA
PCR
Gene X PCR FRAGMENT
No Stress
RT-PCR
Gene X
Transcription
Gene X RNA
AAAAAAAAAAAAAA
RT
Gene X cDNA
PCR
Gene X PCR FRAGMENT
Stress
Cell Cycle
Gene Markers
• CLN2 - G1/S Boundary
• CLB2 - M Phase
Step 1: Experiment
• BAR1 knockout strain (BAR1∆) grown.
• Synchronized.
• Synchronization visualized.
• Synchrony released.
• Time-points: 0, 20, 40, 60, 80, 100 minutes.
•
CLN2
G1 cyclin involved in regulation of the cell cycle; activates Cdc28p kinase to promote
the G1 to S phase transition; late G1 specific expression depends on transcription
factor complexes, MBF (Swi6p-Mbp1p) and SBF (Swi6p-Swi4p).
•
CLB2
B-type cyclin involved in cell cycle progression; activates Cdc28p to promote the
transition from G2 to M phase; accumulates during G2 and M, then targeted via a
destruction box motif for ubiquitin-mediated degradation by the proteasome.
Step 2: RNA Preps
• Time-points: 0, 20, 40, 60, 80, 100 minutes.
0
20
40
60
80
100
Fairchild, 2010
Step 3a: RT-PCR
How do I know if this
is the correct result?
0
20
40
60
CLN2
80
100
Fairchild, 2010
Step 3a: RT-PCR
0
20
40
60
80
100
CLN2
Fairchild, 2010
Step 3b: RT-PCR
0
20
40
60
80
100
CLB2
Fairchild, 2010
Experiment
Team
Heat Stress
Michael Luo
DNA Damage
Katie, Allison
Cold Stress
David
H 2O 2
Michael Z’s
Hyper-osmotic Stress
Wonder Twins
Hypo-osmotic Stress
Wonder Twins
Amino Acid Starvation
Neha, Elise
Experiment
RT-PCR Gene
Expectation
Heat Stress
HSP90
Activation
DNA Damage
RAD54
Activation
Cold Stress
HSP90
Repression
H2O2
CLN2
Repression
(20 minutes)
?
?
?
?
?
?
Hyper-osmotic
Stress
Hypo-osmotic
Stress
Amino Acid
Starvation
Heat Stress Experiment
• Overnight
• Log Growth
• Overnight
• Log Growth
• No Stress
• Snap Freeze
• Heat Stress
• Snap Freeze
from OD 0.2
to ~ 0.8
from OD 0.1
to ~ 0.8
Heat Stress
• Pre-warm media to 39C in carefully
controlled 39C water bath.
• Spin cells down (3000 rpm, 2 min).
• Remove 30C media, re-suspend cells
quickly in 39C media (15, 50 mL conical).
• Place cells (sealed) in 39C water bath for
15 minutes. Mix every few minutes.
• Spin cells down (3000 rpm, 2 min).
• Pour off media & snap freeze cells.
RT Kit
Order of Operations
• Overnights
• Plate Assays (normalized)
• Stress Experiment(s)
• RNA Preps
• RNA Validation
• RT
• PCR on RT-PCR Targets & ACT1
Next Week
• Status on Plates, Experiments
• Computational Process