Wed. 7th
|
Day
1 - Basic qPCR
|
13:00-14.00
|
Basic PCR and qPCR theory and
applications
- Amplification
and detection
- Detection
chemistries
-
Selected applications
|
14.00-15.00
|
qPCR experiment
by participants
- Display of various instrument platforms
- Demonstration of qPCR software
- Practical considerations when preparing PCR reactions
- Programming qPCR machines
|
15.00-15.15
|
Coffee Break
|
15.15-16.00
|
Primer and probe design and
considerations
- What
does primer design affect?
- What
are primer dimers?
- How
do we minimize formation of primer dimers?
- Design
of Molecular Beacons and TaqMan probes
|
16.00-17.00
|
Data analysis
- How
does qPCR software process the data?
- How
are standard curves used and created?
- How
are melt curves used?
- Principle
of quantification using standard curves
- Principle
of relative quantification
|
17.00-17.30
|
Analysis of performed qPCR experiments
|
17.30-17.45
|
Discussion and Q&A
|
17.45
|
End of qPCR workshop day 1
|
|
|
Thu. 8th
|
Day
2 - Advanced qPCR. Quantification, Normalization and
experimental design
|
09.00-09.50
|
qPCR quantification strategies
- standard
curves
- relative
quantification
- how
to compensate for inhibition in biological samples
|
09.50-10.15
|
Normalization of qPCR data
- What
levels of normalization can be used?
- How
to choose a good reference gene?
|
10.15-10.30
|
Coffee Break
|
10.30-11.45
|
Experiment
comparing different quantification strategies
- relative and standard curve quantification
- different efficiency calculations/assumptions
|
11.45-12.45
|
Lunch
|
12.45-13.30
|
Optimization of qPCR protocols
- What
parameters can/should be optimized?
- An
optimization strategy
|
13.30-15.00
|
Quantification calculation
examples
-
what effect will efficiency have on quantification
-
quantification methods, and equations
|
15.00-15.15
|
Coffee Break
|
15.15-16.45
|
Analysis of
experimental data
- differences in quantifications strategies
- effect of efficiency estimations on results
- calculations of relative abundance of genes
- pros and cons of different methods
|
16.45-17.00
|
Discussion and Q&A
|
17.00
|
End of qPCR workshop day 2
|
|
|
Fri. 9th
|
Day 3
- Advanced qPCR: Sample Preparation and reverse transcription
|
09.00-10.00
|
Principle of RT and different
RT priming strategies
- Pros
and cons of different methods
|
10.00-10.45
|
Principle of RNA and DNA extraction
- How
it works
- Available
methods and products suitable for qPCR
- Practical
considerations
|
10.45-11.00
|
Coffee Break
|
11.00-11.45
|
Reverse transcription
experiment using different priming methods
- Oligo(dt)
- Random Hexamers
- Gene specific primers
|
11.45-12.45
|
Lunch
|
12.45-13.30
|
qPCR experiment
evaluating RT using the generated cDNA
- Is there a best RT priming method?
|
13.40-14.30
|
Quality Control in qPCR using
Kinetic Outlier Detection
- How
to detect samples with significant inhibition
|
14.30-14.45
|
Coffee Break
|
14.45-15.30
|
SNP detection. Multiplexing
possibilities and problems
- qPCR
for SNP/mutation detection. What alternatives are there?
- Multiplex
optimization
|
15.30-16.15
|
Analysis of
experimental data
- Which priming method for RT is best?
- How should experiments be planned to take RT priming
into consideration?
|
16.15-16.30
|
Probes and Dyes
-
What dyes/quenchers are typically used in qPCR
-
How to measure the maximum fluorescence available in a dual-labelled probe
|
16.30-16.45
|
Discussion and Q&A
|
16.45
|
End of qPCR workshop day 3
|