Hands-on qPCR

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:

Day 1

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.

 

Day 2

Normalization

  • 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.

 

Day 3

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?

Group discussion

  • 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.