Single-Cell Gene Expression Analysis

The analysis of transcripts in minute samples is often used to increase the knowledge in areas such as cancer development, plant physiology, neurodegenerative disorders of developmental biology. A very precise sampling crucial is crucial for the success of the approach. Depending on the nature of the sample and the goal of the experiment, there are different ways of sampling the probes. This course will present flow cytometry and laser capture microdissection as methods to obtain single cells for the subsequent gene expression analysis. The need for quantitative single-cell mRNA analysis is given by the inability of conventional RNA methods, to differentiate mRNA abundance of such limited samples. Another crucial step is data analysis, which constitutes another strong component of the course.

This course targets scientists who are entering the field of single cell analysis and want to learn about different preparation methods and gene expression profiling. The participants will learn about the complete workflow and gain understanding about the possibilities and limitations of the approach. The course will cover lectures and hands-on sessions by experts in the fields and is aimed for PhD students, postdoctoral fellows or other scientists with a solid qPCR and molecular biology background.
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(Preliminary) Programme:

Day 1 - Saturday 29 June 2013
09:00-21:00
Session 1- Introduction to single cell analysis sampling methods. Chair: Jacqueline Dreyer
Jacqueline Dreyer - EMBL Heidelberg Lecture - Welcome & Introduction: "Breaking the ice"
Anders Ståhlberg - University of Gothenburg Lecture - Introduction to single cell analysis- applications, advantages & pitfalls
Coffee break
Shirley Chu - Life Technologies Lecture - Sampling Methods - LCM (different applications general considerations etc)
Claudia Bevilacqua - INRA Lecture - LCM application
Lunch
Alexis Perez Gonzales - EMBL Heidelberg Lecture - Sampling methods- Flow Cytometry
Alexis Perez Gonzalez -EMBL Heidelberg, Shirley Chu - Life Technologies Practical - Demo sessions for LCM and FC (breakout in groups)
Coffee break
Jacqueline Dreyer - EMBL Heidelberg
Group activity - Expectations
Dinner
Poster session with presentations (19:00-21:00): all – Project presentations
Wine & Cheese

Day 2 - Sunday 30 June 2013
09:00-21:00 Session 2 Lecture - Sampling and qRTPCR/ Fluidigm
Chair: Anders Ståhlberg
Anders Ståhlberg - University of Gothenburg Outline of the day
Christoph Merten - EMBL Heidelberg Lecture - Microfluidics & single-cell gene expression analysis: state-of-the-art
Ken Livak - Fluidigm Corporation Lecture - Fluidigm
Coffee break
Alexis Perez Gonzales - EMBL Heidelberg Practical - Sampling by flow cytometry & cell lysis
Lunch
Anders Ståhlberg - University of Gothenburg Theory - Setup of Reverse Transcription
Vendula Rusnáková - Institute of Biotechnology Practical - Reverse Transcription
Anders Ståhlberg - University of Gothenburg Theory - Single Cell qRTPCR
Coffee break
Vendula Rusnáková - Institute of Biotechnology, Anders Ståhlberg - University of Gothenburg Group activity - Main learning objectives, comparison of the different sampling methods, opening up questions for the next day practical
Dinner
Poster session with presentations (19:00-21:00): all – Project presentations

Day 3 - Monday 1 July 2013

09:00-17:30 Session 3 qRTPCR Chair: Vendula Rusnáková
Vendula Rusnáková - Institute of Biotechnology Lecture - Outline of the day
Vendula Rusnáková - Institute of Biotechnology, Anders Ståhlberg - University of Gothenburg Practical - Single Cell qPCR experiment
Coffee break
Anders Ståhlberg - University of Gothenburg Lecture - Optimisation of a single-cell qPCR experiment
Lunch
Birgit Liss - University of Ulm Lecture - Quantitative RT-qPCR of individual neurons in disease states Practical - Setup of Fluidigm run
Coffee Break (as incubation time permits)
Free Evening

Day 4 - Tuesday 2 July 2013

09:00-20:00 Session 4 Single-cell RNA Seq Chair: Vladimir Benes
Vladimir Benes - EMBL Heidelberg Lecture - Outline of the day
Rickard Sandberg - Lecture - Next Gen Seq, Single-cell RNA Seq: State-of-the-art, applications, advantages & pitfalls
Coffee break
Rickard Sandberg, Paul Collier EMBL Heidelberg Practical - Sample preparation for RNA Seq,
Lunch
Lecture - BioRad- digital PCR during incubation times
Mikael Kubista - Institute of Biotechnology Lecture - MIQE Guidelines and their impact on single-cell qRTPCR
Coffee Break
Mikael Kubista - Institute of Biotechnology Lecture - Data normalisation
Rickard Sandberg, Paul Collier EMBL Heidelberg Practical - Sample preparation and Sequencing run
Dinner

Day 5 - Wednesday 3 July 2013

09:00-20:00 Session 5 Data Pre-processing and statistical analysis
Chair: Mikael Kubista
Mikael Kubista - Institute of Biotechnology Lecture - Outline of the day
Michael Pfaffl - Technical University Munich Lecture - Quantification strategies and efficiency determination
Coffee Break
Anders Ståhlberg - University of Gothenburg, Vendula Rusnáková - Institute of Biotechnology Practical - Missing Data, data normalisation
Lunch
Mikael Kubista - Institute of Biotechnology, Anders Ståhlberg - University of Gothenburg, Vendula Rusnáková - Institute of Biotechnology, Michael Pfaffl - Technical University Munich Practical - Basic analysis, distributions
Coffee Break
Mikael Kubista - Institute of Biotechnology, Anders Ståhlberg - University of Gothenburg, Vendula Rusnáková - Institute of Biotechnology, Michael Pfaffl - Technical University Munich Panel Discussion - Checking and evaluating the outcome with white-board session to assess the critical steps, do’s and dont’s of data analysis
Dinner

Day 6 - Thursday 4 July 2013

09:00-20:00 Session 6 Data Analysis & troubleshooting Chair: Michael W. Pfaffl
Michael Pfaffl - Technical University Munich Lecture - Outline of the day
Mikael Kubista - Institute of Biotechnology, Anders Ståhlberg - University of Gothenburg, Vendula Rusnáková - Institute of Biotechnology, Michael Pfaffl - Technical University Munich Practical - Data analysis cont. - classifications
Coffee Break
Michael Pfaffl - Technical University Munich Lecture - Different relative quantification methods
Lunch
Mikael Kubista - Institute of Biotechnology, Anders Ståhlberg - University of Gothenburg, Vendula Rusnáková - Institute of Biotechnology, Michael Pfaffl - Technical University Munich Practical in groups of 2
Mikael Kubista - Institute of Biotechnology, Anders Ståhlberg - University of Gothenburg, Vendula Rusnáková - Institute of Biotechnology, Michael Pfaffl - Technical University Munich Panel Discu ssion - Q&A – Troubleshooting
Course Dinner Downtown

Day 7 - Friday 5 July 2013

09:00-16:00 Session 7 Fluidigm and RNASeq Data Analysis Chair: Rickard Sandberg
Rickard Sandberg Lecture - Outline of the day Fluidigm Data Analysis
Coffee Break
Demo/Practical
Lunch
Rickard Sandberg Lecture - RNA Seq Data analysis
Demo/Practical
Wrap-up & conclusions - Best poster award

29 Jun - 5 Jul 2013
Heidelberg
Germany
meeting website