Principal Investigators Meetings
Vertical Array Project
Rosana Risques, Martin Judex, Gaelle Rhondeau, John Welsh, Michael McClelland, Charles Berry, Thomas Werner, Miguel Peinado
Table of Contents:
- Title and Author
- Vertical array strategy
- RNA Arbitrarily Primed PCR.RAP-PCR to make LCRs
- RAP-PCR is robust
- Rot curve
- Different RAP probes (red) vs. oligo dT probe (green)
- Oligo dT vs RAP
- RAP vs RAP
- Rap vs. QPCR
- Cumulative Coverage of 3800 Genes
- Comparison with Affymetrix GeneChip
- RAP-array cassette
- Vertical Array Design
- Vertical Array Throughput
- Actin vs. Thrombospondin
- Actin vs. Cyclophilin
- Actin vs. Cyclophilin Ratios
- Technical notes
- Uses of vertical arrays
- Transcription factor prediction
- Experiment size
Vertical Array Project
Sidney Kimmel Cancer Center, San Diego
Coverage of thousands of experimental variables using microarrays is expensive and requires thousands of array hybridizations.
QPCR is great for thousands of variables, but can become costly when hundreds of genes must be examined.
"Vertical arrays" are modified dot blots that are economical when thousands of variables and hundreds of genes are to be examined.
The choice between standard “horizontal” arrays, QPCR, and vertical arrays is a matter of cost benefit analysis
Vertical Array Strategy
- Make low complexity representations of the mRNA populations.
- Spot these on a glass slide.
- Probe one gene at a time, or a few using multichannel fluorescence.
RNA Arbitrarily Primed PCR RAP-PCR to make LCRs
RAP-PCR is robust
These are at a disadvantage because their low sequence complexity results in only a few poor matches.
RAP + Array
The RAP-PCR reactions, themselves, are analyzed by hybridizing to a cDNA array.
Different RAP probes (red) vs. oligo dT probe (green)
Oligo dT vs RAP
RAP vs RAP
RAP vs. QPCR
Genes selected from serum-starvation re-feeding experiment.
Cumulative Coverage of 3800 Genes
(5 best re-sampling average = 1.7)
Comparison with Affymetrix GeneChip
|Stdev above background|
|Genes in common||530||471||378|
|Present in GeneChip||312||291||231|
|% missed by GeneChip||41||38||39|
|Corrected for library errors*||20||19||19|
|*The procedure enriches for library errors.|
RT-PCR across splice junctions confirmed presence of 7 out of 10 messages detected by RAP but undetected by Affy. 3 remaining have not yet been sequenced-confirmed.
(for multiple RAP hybridizations)
Vertical Array Design
Each experiment is represented by ~10-20 spots made using RAP-PCR.
Vertical Array Throughput
Each gene is represented in at least one spot per experiment
Three genes at a time can be studied in thousands of experimental contexts.
Actin vs. Thrombospondin
Fibroblasts from 23 patients and 20 primers at 2 RNA concentrations + controls.
Actin vs. Cyclophilin
(different actin probe)
Actin vs. Cyclophilin Ratios
- Two-color comparison cannot be implemented in the usual way. Multiple randomly selected genes will be used to calibrate the amount of hybridizable DNA in a spot. Possibly in pools.
- Redundancy in data occurs because LCRs overlap.
- Number of RAPs per condition depends on required coverage of exemplar set. Estimate ~20 for 98%.
Uses of Vertical Arrays
- Transcription Factor Predictor: Classification of exemplars, and co-classification of novel cases using vertical arrays to survey thousands of pleiotropic agents or cell lines
- Drug screening: Survey of the impact of thousands of drugs on hundreds of genes.
- Large sample screening: Survey hundreds of genes in thousands of pathology samples.
- Experiment Archive: Keep representation of mRNA populations from many diverse experiments and laboratories for unplanned discovery as insights evolve.
Transcription factor prediction
- Use transcription factor target ‘exemplar’ genes and experimental perturbation using drugs and/or different cell lines to build a model of exemplar gene behavior.
- Select perturbations or cell lines that best distinguish the exemplars from non-exemplars.
- Use these conditions in standard microarrays to discover targets for the transcription factor.
|Table 3: Agents Reported to Alter AP-1 or NF-kappaB controlled genes|
|Agent||AP1 Effect||AP1 Ref.*||NF-kB Effect||NF-kB Ref.**||Agent||AP1 Effect||AP1 Ref.*||NF-kB Effect||NF-kB Ref.**|
|*See References for AP-1 agents in Table 3 in Literature Cited|
|**See References for NF-kappaB agents in Table 3 in Literature Cited|
(for single cell line)
|Pairwise combinations:||33!/2!31! + 33 = 561|
|RAPs per experiment:||10|
|Hybridizations:||108 genes + 20 control genes|
|Standard array equiv:||2244 hybridizations|
|Q-PCR equivalency:||242,352 reactions|
Biomek FX robot to set up reactions, GeneMachines robot for spotting.