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Alfred Wegener Institute (AWI): Workshop on real time quantitative PCR

8 Jan 2019

Workshop on real time quantitative PCR to develop climate services for monitoring harmful microorganisms in their marine environment

21th to 25th October 2019 at Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany

This workshop is aimed at giving participants an understanding of real time quantitative PCR and its applications in harmful algal bloom (HAB) monitoring. The course includes presentations, discussions, and hands-on demonstrations and covers all aspects of qPCR development and application, from designing a qPCR assay and preparation of defined samples to data analysis and trouble shooting. SYBR green and TaqMan assays will be applied with focus on key species groups of HAB microalgae (e.g., Alexandrium, Ostreopsis, Phaeocystis and Pseudo-nitzschia spp.). After completing the course participants will be able to design qPCR assays specific for their individual needs and perform qPCR experiments as well as to interpret and analyze the gained data. Furthermore, the workshop will give insights into fluorescence in situ hybridization (FISH) as a validation tool for quantitative real time PCR.

The workshop is intended for persons working in or planning to initiate harmful algal monitoring programs, and are interested in and equipped to applying this molecular technology. Participation is limited to 10 participants, with a documented professional interest in phytoplankton identification by molecular biological methods. Applications should be sent by email to: Kerstin Klemm (Kerstin.Klemm@awi.de; cc: Allan.Cembella@awi.de, Uwe.John@awi.de, Raffaele.Siano@ifremer.fr) before April 15, 2019. A short letter justifying the participation of the applicant and details of the proposed application of the knowledge gained to HAB monitoring should be submitted. A brief CV of the applicant’s experience and qualifications must also be provided. The course will be taught in English and a good knowledge of English is therefore required. There will be no registration fee, but participants will have to provide for their own travel and accommodation expenses.

Goals of the Workshop

  • To give participants a deeper understanding of qPCR and its applications
  • To improve harmful algal bloom detection by using molecular tools
  • To explore the potential application of qPCR for the monitoring and forecasting of harmful algal blooms in (own) monitoring programs
  • To provide guidance for developing own qPCR assays for desired target species
  • To validate qPCR achieved results by applying light microscopy and fluorescence in situ hybridization (FISH) on selected samples

Workshop agenda

1st day

Monday, 21 October 2019

 

 

9:00- 9:10

Welcome and Workshop Overview 

 

Uwe John, Kerstin Klemm, Raffaele Siano, Allan Cembella

9:10-9:30

Presentation of the hosting institute and introduction of workshop attendees

Uwe John

 

 

9:30 - 10:00

Theoretical session

Introduction to project CoCliME

Harmful algal blooms, and

coastal ecosystem status indicators in the project CoCliME

Kerstin Klemm

10:00-10:30  

Theoretical session

 

Introduction to PCR:                            

a general overview

  • Qualitative PCR and quantitative PCR, theory and applications
  • qPCR platforms, amplification and detection

Detection chemistries

Uwe John

10:30-10:45

Recreational break

 

10:45-11:15 Theoretical session

 

Different sampling methods of field samples

Quantitative PCR applications in detecting harmful microalgae and their use in HAB monitoring programs

with focus on the HAB species involved in monitoring programs of  CoCliME endusers

 

Raffaele Siano, Kerstin Klemm

11:15-12:00  Theoretical session

 

Primer and probe design for qPCR applications

General overview:

  • Probes and dyes
  • What does primer design effect?
  • What are Primer Dimer formations? How could we minimize it?
  • Design and specificity testing of primer and probes

Uwe John

12:00-13:00

Lunch at AWI canteen

 

13:00-17:00

Practical session

 

  • Microscopic counts of cells
  • Extraction of DNA of different cell numbers, extraction of DNA from (own) field samples 
  • Qualitative PCR, preparation of qPCR samples and preparation of standard dilution series

Kerstin Klemm, Uwe John, Raffaele Siano

17:00

End of Workshop day 1

 

2nd day

Tuesday, 22nd October 2019

 

 

9:00 – 09:30       

Theoretical session

  • Creation of standard curves with genomic DNA or plasmid DNA templates
  • Standard curve quantification in qPCR approaches:
  • absolute and relative quantification
  • Inhibition in biological samples and how to compensate

Uwe John

9:30-9:45

Recreational break

 

9:45-12:00

Practical session

 

  • Demonstration of qPCR instrument and qPCR software  
  • Demonstration of setting up a qPCR experiment
  • Setting up a qPCR experiment by participants:
  • Preparation of standard curves and samples, preparation of positive and negative controls

Kerstin Klemm, Uwe John

12:00-13:00

  • Lunch at AWI canteen

 

13:00-14:30

Practical session

Data analysis

  • How does Step one plus software process the data?
  • How are melt curve used?
  • How do we use standard curves for quantification?
    • Quantification methods and equations
    • Differences in quantification strategies
    • Advantages and disadvantages of methods
    • Which effect will assay efficiency have on quantification?

Kerstin Klemm, Uwe John

14:30-17:00 

Practical session

Analysis of performed  qPCR experiments

Kerstin Klemm, Uwe John, Raffaele Siano,

17:00

End of Workshop day 2

 

3rd day

Wednesday, 23 October 2019

 

 

9:00 – 12:00                         

qPCR analysis of field samples using SYBR Green or TaqMan assays

Kerstin Klemm, Uwe John, Raffaele Siano

12:00-13:00

Lunch at AWI canteen

 

13:00-17:00

End of Workshop day 3

 

4th day

Thursday, 24 October 2019

 

 

9:00-9:15

Theoretical session

 

Introduction to alternative analysis methods in detecting harmful algal blooms with focus on fluorescence in situ hybridization techniques

Kerstin Klemm

9:15-12:00

Practical session

Practical demonstration of FISH experiments and analysis of data

 

Kerstin Klemm, Uwe John

12:00-13:00

Lunch at AWI canteen

 

13:00-17:00

Combined theoretical and practical session

Further qPCR analysis of

field samples, analysis of performed qPCR experiments

Kerstin Klemm, Uwe John, Raffaele Siano,

17:00

End of Workshop day 4

 

5th day

Friday, 25 October 2019

 

 

9.30-12:00

Combined theoretical and practical session

 

Ongoing analysis of performed qPCR experiments, optimization  strategies,

trouble shooting

  • Future plans, questions and answers
  • Workshop summary

Kerstin Klemm, Uwe John, Raffaele Siano

12:30

End of workshop