Transcript ASU_TVDC_ tech call minutes_04-28-2009 final

ASU TVDC Progress Report
4/28/09
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25, 26, 28 and 32, 33, 34
Active Milestones: 35, 36
Currently Inactive Milestones: 30, 37, 38
Slide 1
MILESTONE 35
Array hybridations with mouse RNAs
from virulent Schu 4 infection
& RT PCR confirmation of candidates
Gray: (sub )milestone title
Red: completed
Green: in progress
Virulent Schu 4 Samples
RT-PCR Confirmations
Initial samples
Dose-Response of Infection
Ongoing
Slide 2
Current Status
• Overcome the LAPT amplification problems
– Have LAPT amplified two mouse time course and 1 rat time course samples
• qPCR determinations
– iglC quantifiable signals detected to between 200-500 bacteria per lung
– MutS was not detectable until > 1-2000 bacteria per lung
• Low-level signal intensities
– Slide problems (age of slides / probe questions)
– Technical issues solved
Slide 3
Labeled
Un-Labeled
Bioanalyzer Analysis of Labeled cDNA
Slide 4
Slide 5
Raw Signal Intensity
Changing Hybridization Conditions
Static
Agilent
Slide 6
Raw Signal Intensity
Scanner Effects
200 pM cDNA
10 pM G
200 pM cDNA
20 pM G
200 pM cDNA
40 pM G
Slide 7
Conclusions
• Lower Signal Intensities
• Technical issues (hybridization temp)
• Scanner problems
• Scanner repaired
•New backup scanner on order
• Use Agilent chambers over static chambers
Slide 8
MILESTONE 36
Final integration of expression data
and informatics analysis
Gray: (sub )milestone title
Red: completed
Green: in progress
Ongoing
Slide 9
Upcoming Transcriptome Goals
• LAPT
• 2 New Mouse Experiments (> 1-2000 CFU at T1)
• 1 New Rat Experiment
• Gene Selection and qPCR verification
• Final 10 candidates selected
Slide 10
Action Items
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Should ASU switch to Agilent to increase signal intensity on future hybs? ASU is
considering switching to the Agilent system for hybridizations.
UNM/Terry Wu: will send more infected mouse and rat lung RNAs to ASU in mid May
2009. Terry and Mitch have decided on the details, but higher CFU levels starting at
the first time point are needed (1000-2000 CFU ).
ASU will compare the gene lists for static, increasing, and decreasing expression,
between mouse and rat species.
ASU may have the mouse and rat gene expression data to share by early June.
Thereafter, UNM, ASU and others could have a discussion of a subset of
approximately 10 genes in June.
NOTE ADDED AFTER ASU 4/28/09 TECH CALLUNM and ASU are planning an ad
hoc call to discuss the reanalysis of the first round polypeptide/ELIspot data and the
conservative re-screening of a few positives using the frozen , vaccinated NHP
splenocytes/lymph node cells. (Scheduled for 5/8/09).
Slide 11