Transcript ASU_TVDC_01-27-2009 minutes

ASU TVDC Progress Report
1/27/09
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25, 26, 28 and 32, 33, 34
Active Milestones: 35
Currently Inactive Milestones: 30, 36-38
Slide 1
MILESTONE 28
Build SCHU S4
proteome
Gray: (sub)milestone title
Red: Complete
Build ORF expression
library corresponding
to proteome
Generate complete
protein-fragment library
Array protein-fragments
for T cell stimulation
assays
Complete
Complete
Complete
Slide 2
Diagram for pooling distribution
FTU IVT Pool
Round 1
Plate 1
360ul
FTU IVT Pool
Round 1
Plate 1
180 ul
(For Lymphocyte assay)
FTU IVT Pool
Round 1
Plate 1-1
90 ul
FTU IVT Pool
Round 1
Plate 1-2
90 ul
FTU IVT Pool
Round 1
Plate 1
180 ul
(For PBMC assay)
FTU IVT Pool
Round 1
Plate 1-3
90 ul
FTU IVT Pool
Round 1
Plate 1-4
90 ul
Plate arrangement of FTU IVT proteins
Plates
Sources of FTU IVT proteins
FTU IVT Pool
Plate 1
Short ORF1, Short ORF 2, and Long ORF 5
FTU IVT Pool
Plate 2
Long ORF 4 and Long ORF 3
FTU IVT Pool
Plate 3
Long ORF 2 and Long ORF 1
FTU IVT Pool
Plate 4
14 FTU IVT proteins from Short ORF 2 and all samples from BAG
plates
Summary of FTU library
1. 2,229 FTU proteins were successfully
translated in vitro and arrayed into 319
pools
2. Each pool consists of 7 different proteins
3. Only half of IVT reaction was sent to
UNM and used for T-cell assay
4. The remaining half is stored at ASU for
future experiment
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 6
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 > 2000 bacteria per lung
• Low-level signal intensities on latest batches of arrays
– Questioned whether the age of the slide was a problem
Slide 7
Raw Signal Intensity
Histogram of Microarray Signals of Rat Timecourse LAPT
0
1
3
5
7
24
Hours Post Challenge
Slide 8
Rat Time Course
Slide 9
Raw Signal Intensity
Histogram of Microarray Signals of SCHU S4 RNA
Historical
6-Month
Old Slide
New Print
Slide 10
Upcoming Transcriptome Goals
• LAPT
• All samples have been amplified
• Microarrays
• Perform titration analyses to verify loading
• Make new master plates
•qPCR
• Run other gene sets with samples with
quantified bacterial numbers.
Slide 11
Action Items
• No new action items, other than ASU’s
planned experiments and troubleshooting
Slide 12