Transcript ASU_TVDC_ tech_call_06-23-2009 minutes final

ASU TVDC Progress Report
6/23/09
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25, 26, 28 and 32, 33, 34
Active Milestones: UNM 29 and ASU 35 &36
Currently Inactive Milestones: 30, 37, 38
Slide 1
UNM Milestone 29 – flow diagram
SOP for detecting T cell
stimulation with ivt
proteins and peptide
Production of ivt
proteins &
peptide library
(ASU)
Production
Blue: Steps in the milestone
Red: Completed
Green: In progress
Assay
development
(UNM)
T cell proliferation
IFNg ELISpot assay
Screening
(UNM)
Identification of
stimulatory proteins &
peptides
Confirmation
Assay optimization
using ivt proteins
2
Elispot Stats
• Analysis technique: Original data is from Lymph node and Splenocytes
across 4 replicate plates and across 4 sets.
• Analysis was population based, looking at any value that significantly
exceeded the mean of that population
• Original analysis used mean + x standard deviations
• New analysis used 2 methods: power calculation and confidence intervals.
Slide 3
Elispot Stats
• Power analysis determined the minimum detectable differences
• Selections were identified by adding the minimum detectable
difference (delta) to the mean and selecting any value that exceeded
the mean + delta
• Minimum detectable difference was calculated per population.
Values that exceeded delta were considered significant at 95%
confidence and 80% power.
Slide 4
Elispot Stats
• New analysis also used a calculation of confidence intervals at 99%ile.
• The mean of the sample + the 99% CI indicates that the observation
exceeded the mean by a significant amount.
• CI = avg. +/- 2.56 (stdev/sqrt(n))
• As it turned out, the power analysis was the most conservative…
• Since so many wells would have been selected using delta as a minimum
cutoff, I estimated the highest difference, highest reproducibility, and best
confidence interval and power to get a small set of “high and
reproducible” wells to re-test.
Slide 5
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
MILESTONE 36
Final integration of expression data
and informatics analysis
Gray: (sub )milestone title
Red: completed
Green: in progress
Ongoing
Slide 7
Current Status
• Two new (high dose) mouse and 1 second rat RNA prep received
from UNM
• Two LAPT processes run for each of the mouse (both high and lowdose experiments
• One labeling and hybridization from high-dose mouse experiment
completed
• Compared data to the two low-dose mouse experiments
• All data from Agilent-hybridized arrays using genomic normalization
• Pattern analysis picking the top 200 gene patterns
– Up over time
– Down over time
– Flat over time
Slide 8
Current Status
Design
Low-Dose Mouse
Sample Sets
1
LAPT
Slides
1
High-Dose Mouse
2
2
1
1
2
1
Rat
2
2
1
1
2 1
2
2
1
2
Red = completed
Green = ongoing
Slide 9
Genomically-Normalized Signal Intensity
Pattern Mapping
Up
Flat
Down
0
1
3
5
7
24
Slide 10
Mouse Low-Dose Challenge
Down
Flat
116
86
100
0
0
14
185
Up
Slide 11
Mouse High-Dose Challenge
Down
Flat
128
71
47
2
0
82
115
Up
Slide 12
Rat Challenge
Down
Flat
167
38
83
0
0
80
118
Up
Slide 13
Flat-Restricted
Mouse Low-Dose
(100)
Mouse High-Dose
(47)
46
0
93
0
1
7
FTT0547
Rat
(83)
75
FTT0294
FTT1612
FTT0551
FTT0758
FTT0493
FTT1460c
FTT0517
Slide 14
Down-Restricted
Mouse Low-Dose
(116)
Mouse High-Dose
(128)
FTT0643
FTT1160c
FTT1189c
FTT1161
FTT1491c
FTT0973
FTT1159c
FTT1577
FTT0281
FTT1050c
FTT0205
FTT0795
FTT1210c
FTT0754c
FTT1563
FTT0809c
FTT0909
FTT1248
FTT0156
109
8
88
1
11
Rat
(167)
19
136
FTT0002
FTT0117
FTT0440c
FTT0943c
FTT0427
FTT0111
FTT1122c
FTT0302
FTT1070c
FTT1602
FTT0345
FTT0123
FTT1404
FTT1639c
FTT0726c
FTT1572c
FTT1270c
FTT0601
FTT0695
FTT0241c
Slide 15
Up-Restricted
Mouse Low-Dose
(185)
Mouse High-Dose
(115)
103
10
FTT0864c
FTT1085
FTT1504
FTT0413c
FTT1170
FTT1345
FTT0661c
FTT0198
FTT1415
FTT0802
169
0
2
6
FTT1030
FTT0303c
Rat
(118)
110
FTT1239
FTT1207
FTT0876c
FTT0410
FTT0828c
FTT1559c
Slide 16
Upcoming Transcriptome Goals
• LAPT
•1 New Rat Experiment
• Label, hybridize last samples
• Gene Selection and qPCR verification
• Select 10 candidates
Slide 17
Action Items
•
•
•
UNM will proceed with retesting a subset of the positives identified by Phil’s
Elispot analysis of the full proteome library screening.
ASU will compare the ASU microarray gene sets to literature set being built
at ASU and to ASU proteomics data sets
ASU will update UNM and will print more microarray slides by early July
and then QC verify the newly printed slides in July 2009.
Slide 18