Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins
Transcript of Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins
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8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins
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Elective course in Experimental Systems
Analysing gene expression
Classical methods to determine the
expression patterns ofmRNAs and proteins
Dr. Ian [email protected]
Centre for Integrative Physiology&
Institute for Adaptive and Neural Computation
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8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins
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Ian Simpson, University of Edinburgh
The Central Dogma of Molecular Biology
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Ian Simpson, University of Edinburgh
The Central Dogma of Molecular Biology
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Ian Simpson, University of Edinburgh
Gene structure
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Transcription
Ian Simpson, University of Edinburgh
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Alternate splicing
Ian Simpson, University of Edinburgh
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8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins
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Ian Simpson, University of Edinburgh
Translation does not just occur near the nucleus
At remote locations (this is a synapse !)
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8/7/2019 Experimental Systems Lecture 1 - Recent developments in detecting mRNAs and proteins
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Ian Simpson, University of Edinburgh
Proteins can be localised to different compartments
Nuclear(Otx2-GFP)
mitochondrialcytoplasmic
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Ian Simpson, University of Edinburgh
Gene expression is variable and can bemeasured at several levels
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Ian Simpson, University of Edinburgh
Generating probes for nucleic acid hybridisation
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Ian Simpson, University of Edinburgh
Southern and Northern blotting
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Ian Simpson, University of Edinburgh
Southern and Northern blotting
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Ian Simpson, University of Edinburgh
Multi tissue Northern, zoo blot
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Ian Simpson, Centre forIntegrative Physiology, University of Edinburgh
RNA in-situ hybridisation
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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
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Ian Simpson, University of Edinburgh
PCR the polymerase chain reaction
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Ian Simpson, University of Edinburgh
Detecting and quantifying RNA by PCR (RT-PCR)
RNA -> cDNA
(reverse transcriptase)
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Ian Simpson, University of Edinburgh
Quantifying RNA with Real time PCR
detection model of qRT-PCR reaction
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Ian Simpson, University of Edinburgh
Quantifying RNA with Real time PCR
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Ian Simpson, University of Edinburgh
Detecting proteins with antibodies
direct detection
Indirect detection
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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
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Ian Simpson, University of Edinburgh
Detecting Proteins in tissue
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15 MINUTE BREAK
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Analysing gene expression
Recent developments in detectingmRNAs and proteins