qPCR

Note: The Doctoral Schools will not refund a qPCR course followed directly at Outside The Lines.

Topic and theme

During the last decades, the number of researchers from different disciplines performing transcriptome analysis has increased dramatically. Despite the use of state-of-the-art high-throughput technologies for whole genome transcriptome profiling (e.g. next generation sequencing or microarray), quantitative real-time PCR (qPCR) remains the gold standard for targeted gene expression analysis due to its superior sensitivity, specificity, linear dynamic range of quantification and a high level of flexibility. A qPCR experiment involves several steps ranging from experimental design, RNA extraction, assay design, data generation to data analysis. While the practical performance of a real-time PCR quantification experiment is relatively straightforward, it is important to identify critical steps that may impact the quality and robustness of the experiment. Moreover, many researchers experience a genuine need for more in-depth training of their experiment design and data analysis
skills in order to generate biologically relevant and reliable results. To accommodate these needs, Outside The Lines offers a qPCR-focused course in which the fundamental and advanced principles of experiment design, sample and assay quality control and data analysis are covered.

Trainer

dr. Bram De Craene, Outside The Lines (sidelines.eu), has a Master in Biotechnology (2001, UGent) and a PhD in Sciences (2005, UGent). He worked as postdoctoral researcher at the Inflammation Research Center department of VIB, where he was managing the departmental qPCR facility, and is author of several scientific articles in the cancer research field. As consultant for Biogazelle/Cellcarta he worked as trainer for Biogazelle’s qPCR courses and in the technical support for the qPCR data analysis software qbase+. Currently, Bram works as Principal Scientist assay design and development at Biocartis.

Programme

The course comprises different chapters covering the whole qPCR procedure from experimental design to data generation and interpretation of the results. Practical examples and exercises are given for each section, allowing participants to interact and formulate their questions. For each chapter, a balanced mix between theoretical background and practical hands-on data analysis is included. During the course, practical examples using the qPCR data analysis software qbase+ and/or Excel are foreseen.

1. Experiment design

  • Power analysis
  • Experiment layout (sample vs gene maximization)
  • Biological and technical replicates
  • Inter-run calibration

2. Sample preparation and quality control

  • RNA integrity and purity
  • DNase treatment
  • cDNA synthesis
  • Pre-amplification

3. Assay design and validation

  • Primer design
  • Probes vs intercalating dye
  • Design guidelines
  • In silico evaluation
  • Empirical validation

4. qPCR amplification

  • Real-time PCR principle
  • Amplification curve
  • Melt curve
  • Cq value determination methods
  • Replicates and controls
  • Speed and throughput considerations

5. Absolute quantification

  • Standard curves
  • Limitations and concerns

6. Relative quantification

  • Quantification models
  • Efficiency correction
  • Selection and validation of reference genes
  • Normalization with multiple reference genes
  • Alternative normalization methods
  • Inter-run calibration
  • Result rescaling

    7. Data quality and control

    • Visual inspection of plots
    • Positive and negative controls
    • Normalization factor histogram
    • Reference gene stability
    • Inter-run variation

    8. Bio-statistical analysis

    • Basic principles
    • Descriptive statistics
    • Selection and application of appropriate statistical tests

    Dates - Venue

    Dates 

    Time Venue/Room

    13-14 June 2024

    From 9:00 - 17:00 (including several breaks) Konrad Zuse (Campus Sterre, S)

    Registration

    Follow this link for the registration and waiting list.

    Your registration will be confirmed by separate e-mail from the Doctoral Schools. Cancellation of your registration can only be performed by sending an email to doctoralschools@ugent.be.

    Registration fee

    Free for members of the Doctoral School.

    The no show policy applies.

    Evaluation criteria (doctoral training programme)

    100% participation

    After successful participation, the Doctoral Schools will add this course to your curriculum of the Doctoral Training Programme in Oasis. Please note that this takes up to one to two months after completion of the course.

    Course material

    One week before the course, an email will be sent to all attendees providing them with an e-copy of the course and supplemental course material. 

    Please note that the qPCR data analysis software qbase+ runs exclusively on Windows.

    Number of participants

    Maximum 20