Gene expression profiling in single cells

Transcription

1 Gene expression profiling in single cells Anders Ståhlberg Department of Chemistry & Biosciences / Molecular Biotechnology Chalmers University of Technology

2 Outline Technical considerations Gene expression profiling Outline

3 Why single cells? Background

4 Workflow single cell analysis Ce llcollection c olle c tion methods Cell Lysis Patch clamp capillaries Heat Lysis 80 d e g re e s 5 m in Reverse transcription RT e nzym e Flow cytometry RTp rim e rs Detergents (Igepal, Reproducibility RNa se inhib ito r Triton...) Laser dissection RT Buffe r Sufficient cdna yield Physical disruption RT Background RNA Pre-amplification d e g re e s Data analysis QPCR d e g re e s appropiate statistics number of cells Da ta a na lysis Linear amplification at Real-time PCR mrna level > 3h Gene specific assays amplification at Exponential C DNA cdna PCR inhibition level Limited amount PCR m a ste r m ix of with p rim ematerial rs for biological ta rg e t g e ne s Suitable controls Gene Gene expression expression profiling profiling in in single single cells cells Technical Technical considerations considerations

5 Gene expression in single cells Gene expression Cell number

6 mrna distribution Cell count Cell count Relative transcript levels Relative transcript levels Linear scale Lognormal scale

7 The median cell Geometric mean Arithmetic mean Cell count Relative transcript levels Lognormal scale

8 Effect of glucose Ins1 Ins2 20 mm Arithmetic Geometric ActB Ins Ins mm Abcc Kcnj

9 Models of gene expression Without e nha nc e r With e nh a nc e r Gene Gene E ( N N mrna mrna ) total = N mrna = = 6 Cell Rhe o sta tic m o d e ( N mrna N) total mrna = = 0 + N0 mrna = = Cell 6 Bina ry m o d e

10 Distribution skewness Skewness = Skewness = Skewness = 0 Skewness = 0.06 Skewness =

11 Gene regulation at single cell level Arithmetic Geometric ActB Ins Ins Abcc Kcnj

12 Gene regulation -case I Gene 1 Gene 1 Intermediete Low expression Gene 2 expression Gene 2 High expression

13 Gene regulation -case II Gene 1 Gene 1 Intermediete Low expression Gene 2 expression Gene 2 High expression

14 Gene regulation in individual cells ActB Ins1 Ins2 Sur1 Kcnj11 ActB 1 Ins Ins Sur Kcnj

15 Gene regulation - Ins1 & Ins2 Ins1 Ins1 Intermediete Low expression Ins2 expression Ins2 High expression

16 Gene regulation - Ins1 & ActB / Ins2 & ActB Ins1/2 Ins1/2 Intermediete Low expression ActB expression ActB High expression

17 Conclusions The combination of patch-clamp capillaries and reverse transcription realtime PCR is suitable for gene expression studies in individual cells Gene expression is lognormally distributed at a single cell level Gene regulation at single cell level do not necessary correlate at cell population level Bengtsson M. et al. Genome Research, in press Conclusions

18 Acknowledgments Martin Bengtsson Mikael Kubista Patrik Rorsman TATAA Biocenter