This introductory course consists of a theoretical part and a practical part where participants get to do qPCR experiments by themselves under experienced supervision.
Testimonial from previous course participant:
“An excellent course that I would recommend to scientists who are relatively new to qPCR through to experienced end users who could do with some revision + fine tuning.“
There is a 3 day course and a 2 day course available. In the 2 day course the third day is omitted.
2 or 3 DAYS Hands-on qPCR
Target audience: Beginners to Medium experienced qPCR users
Entrance qualifications: Basic Molecular biology or similar
Description: The basic real-time qPCR course. You will aquire a comprehensive overview of the possibilities with real-time PCR, how to use it and how to analyze the results.
The course contains:
Introduction to PCR and qPCR
- How does qPCR work?
- Different detection chemistries, dyes or probes?
- Different applications for qPCR.
qPCR data evaluation
- How does qPCR software process the data.
- How to evaluate curves and set threshold.
Primer and probe design
- How to do proper primer design.
- How to avoid primer dimer formation.
- Other important considerations for primer design.
- How to design hydrolysis probes.
- Practical exercises in primer design.
Optimization of qPCR protocols
- Which factors affect the PCR?
- Which factors can be optimized?
Hands-on lab running qPCR analyzing unknown samples with standard curve.
- Different ways to normalize.
- How to find stable reference genes.
Basic quantification theory
- Quantification methods and equations.
- How to do interplate calibration.
Absolute quantification strategies
- How to do absolute quantification.
- What is a suitable standard?
Validation of assays
- How to determine LOD and LOQ.
- Precision estimation.
- Which controls should be used?
Quantification calculation examples
- Practical examples with relative quantification calculations.
- Calculations with own generated qPCR data.
Hands-on lab running qPCR and calculating relative quantities with different strategies.
Sample preparation and isolation
- What to consider during the sampling process.
- How can nucleic acids be extracted?
- How do we quality control the nucleic acids?
Reverse transcription, RT
- Which priming strategies can be used?
- Efficiency and reproducibility of reverse transcription.
The MIQE guidelines
- Why do we need the MIQE guidelines?
- Which information should be included in publications?
- You are welcome to bring your own questions.
Hands-on lab running RT and qPCR investigating what happens if we have inhibitory samples and how a spike can be used to detect it.