8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

1/23

Elective course in Experimental Systems

Analysing gene expression

Classical methods to determine the

expression patterns ofmRNAs and proteins

Dr. Ian Simpsonian.simpson@ed.ac.uk

Centre for Integrative Physiology&

Institute for Adaptive and Neural Computation

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

2/23

Ian Simpson, University of Edinburgh

The Central Dogma of Molecular Biology

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

3/23

Ian Simpson, University of Edinburgh

The Central Dogma of Molecular Biology

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

4/23

Ian Simpson, University of Edinburgh

Gene structure

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

5/23

Transcription

Ian Simpson, University of Edinburgh

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

6/23

Alternate splicing

Ian Simpson, University of Edinburgh

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

7/23

Ian Simpson, University of Edinburgh

Translation does not just occur near the nucleus

At remote locations (this is a synapse !)

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

8/23

Ian Simpson, University of Edinburgh

Proteins can be localised to different compartments

Nuclear(Otx2-GFP)

mitochondrialcytoplasmic

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

9/23

Ian Simpson, University of Edinburgh

Gene expression is variable and can bemeasured at several levels

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

10/23

Ian Simpson, University of Edinburgh

Generating probes for nucleic acid hybridisation

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

11/23

Ian Simpson, University of Edinburgh

Southern and Northern blotting

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

12/23

Ian Simpson, University of Edinburgh

Southern and Northern blotting

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

13/23

Ian Simpson, University of Edinburgh

Multi tissue Northern, zoo blot

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

14/23

Ian Simpson, Centre forIntegrative Physiology, University of Edinburgh

RNA in-situ hybridisation

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

15/23

Ian Simpson, University of Edinburgh

Whole-mount in situ hybridisation with a Drosophila citron DIG-labelled RNA probe. (E-H) Zygotic tissue-specific expression of citron in theproliferating CNS (arrow) and PNS (arrowhead) starts during germ-band retraction. Anterior is to the left, dorsal to the top.

Whole mount RNA in-situ hybridisation

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

16/23

Ian Simpson, University of Edinburgh

PCR the polymerase chain reaction

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

17/23

Ian Simpson, University of Edinburgh

Detecting and quantifying RNA by PCR (RT-PCR)

RNA -> cDNA

(reverse transcriptase)

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

18/23

Ian Simpson, University of Edinburgh

Quantifying RNA with Real time PCR

detection model of qRT-PCR reaction

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

19/23

Ian Simpson, University of Edinburgh

Quantifying RNA with Real time PCR

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

20/23

Ian Simpson, University of Edinburgh

Detecting proteins with antibodies

direct detection

Indirect detection

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

21/23

Ian Simpson, University of Edinburgh

Detecting Proteins by Western blotting

Protein isolated from cell lysates

ERK - green-fluorescent Qdot 565 F(ab)2goat antimouse IgG

PhosphoERK - red-fluorescent Qdot 655 F(ab)2

goat anti

rabbit IgG

Invitrogenfluorescent Qdot nanocrystal conjugates

Detecting total and phospho-ERK

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

22/23

Ian Simpson, University of Edinburgh

Detecting Proteins in tissue

8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins

23/23

15 MINUTE BREAK

NEXT LECTURE

Analysing gene expression

Recent developments in detectingmRNAs and proteins