analysis of gene expression Flashcards
whats the central dogma
-DNA makes RNA which makes protein:
=replication= DNA->DNA
=transcription= DNA->RNA
=translation= RNA-> protein
-Can measure activity of a gene at different levels:
=Transcription (RNA)
=Translation
=Molecular interactions – protein don’t work alone
what do we need to know for gene expression
-How much is there?
-Where is it?
-What is it interacting with?
what are the different ways to analyse gene expression
-RNA expression and localisation
-Protein expression and localisation
-Analysis of molecular interactions
-There are some common themes – same techniques / molecules:
=DNA / RNA – PCR / Hybridisation-based methods
=Proteins – immunological methods / fusion proteins
RT-PCR methods of gene expression
-quantitative or non-quantitative
-Can be quantitative – relative levels of an RNA in different samples / quantify against a standard
-Or not quantitative – run end products on a gel:
=This can give information about relative amounts
=Or can potentially distinguish isoforms by size/RE digest
what are the Hybridisation-based techniques
-Based on properties of DNA/RNA – complementary sequences will hybridise to each other – DNA/DNA or DNA/RNA
-If you have a sequence that you are interested in (RNA/cDNA/DNA), you can use that sequence to make a “probe”
-Probe = DNA sequence that is complementary to the RNA sequence you want to analyse – labelled in some way that we can analyse (various ways to do this)
-This binds in a sequence-specific manner using base-pairing rules
What would be a probe for this RNA molecule
5’-AUUCGCCUAAGAUCGU-3’
-complement : 3’-TAAGCGGATTCTAGCA-5’
-reverse: 5’-ACGATCTTAGGCGAAT-3’
whats RNA expression: Northern blot
1)RNA is separated by electrophoresis – will see major bands of rRNA (many others not visible by eye or identifiable)
2)RNAs are transferred to a membrane (technical reasons – not going into detail here).
3)Probe – complementary to your chosen sequence – labelled (usually radioactive for detection in phosphorimager):
-Can tell you about abundance, size, isoforms, expression in different tissues/cell types
-Limited to a single gene each time
whats RNA expression: microarrays
-Oligonucleotide = short DNA / RNA molecules (type of probe)
-Spot: lots of the same oligonucleotide (rather than one single oligonucleotide)
-RNA is not very stable – cDNA is a substitute
-Can study of 1000s of genes, simultaneously. Expression profile (RNA) of a particular cell-type / tissue / organ
how do Microarrays measure relative mRNA levels
-Not easy to quantify absolute values of mRNA levels
-Better for assaying relative levels (a bit like qPCR)
-Different fluorescent tags for cDNA from different sources
How do we compare levels of transcription
-If there are equal amounts of RNA from both types of cell, the resultant fluorescence will be yellow (red + green)
-Red/green means that one cell has more of that cDNA (green = tumour; red = normal)
-Gives you info about RNA levels. Can give info about isoforms too, depending on what oligonucleotide is chosen to be the probe
-Hybridisation techniques can also tell us about RNA localisation
whats RNA localisation: Fluorescent in situ hybridisation
-Fluorescent In situ hybridisation (FISH for short)
-Principle of hybridisation is same as for Northern blot
-Probe is labelled with a fluorescent marker and visualised using microscopy
=Localisation of RNA
=Or many RNAs (multiple probes)
=Or RNA and proteins
-Correct RNA localisation can be vital for correct development
-Can also be used to localise DNA
what are reporter genes
-Protein localisation
-To measure expression (RNA level)
what are Common enzymatic reporter genes
-luciferase and beta-galactosidase:
-Clone next to a promoter of interest
-Put recombinant promoter-reporter into cells
-Investigate different growth conditions, role of transcriptional activators / repressors, etc.
-The reporter protein is an easily measurable proxy for promoter activity
-↑ Expression of reporter gene = ↑ promoter activity
what are Protein-specific antibodies
-A lot of protein detection methods use antibodies – immune molecules that recognise specific protein(s)
-Secondary antibody recognises the primary antibody – usually raised to the species (anti-rabbit / anti-mouse)
-Usually conjugated to a molecule that allows detection (specific for application)
-Might sound complicated – allows use of primary Abs in lots of applications
whats western blotting
- Proteins are separated by electrophoresis (based on size)
- then transferred to a membrane
- Detection is using a protein-specific antibody and a labelled secondary antibody
-Can tell us about protein levels; isoforms (if they’re different sizes); sometimes PTMs, expression patterns in different cells/tissue
whats immunofluorescence
-Secondary antibody is usually conjugated to a fluorescent molecule – imaged using microscopy
-Use different fluorophores – can see more than one molecule at a time
-Not live imaging – a snapshot in time (cells usually need to be fixed and prepared in quite harsh ways)
how does Live imaging of protein localisation occur
-Use molecular cloning:
=Fuse the CoDing Sequence (CDS) for your protein of interest (Protein X) to that of a fluorescent protein (GFP)
-Fluorescent proteins can be directly visualised
(Other fusion proteins exist for different applications)
how can you combine different techniques while localising proteins
-GFP as a reporter gene for one protein
-And an antibody to visualise another protein
-Shows colocalisation of the two proteins – this may be functionally significant
what are some Tools for analysing interactions
-Proteins rarely function alone. They usually function as part of large complexes with other molecules
-Identifying a protein’s interacting partners might be very useful functional information
-Protein-protein interactions:
=Pull-down assay
=Immunoprecipitation
=Yeast two-hybrid
-Protein-DNA interactions:
=Chromatin Immunoprecipitation
whats Pull-down assay
-A way of analysing protein interactions in vitro
-Uses fusion proteins
=Affinity for a specific ligand -> easy to purify
=Glutathione S-Transferase (GST) is commonly used
=Binds reduced glutathione (GSH)
-Make a recombinant DNA molecule
-GFP was a fusion protein -our fusion protein has affinity for something so we can purify it (GST binds GSH)
E. coli as a factory to make proteins
-Recombinant DNA ->recombinant protein
-Bacteria can make lots of recombinant protein for us – if you give them the right recombinant DNA
-(Fusion protein – one polypeptide chain from one mRNA transcript. The “fusing” happened when you made the recombinant DNA)
what are the steps in pulldown assay
1)Make a cell lysate
2)Bind GST (+ Protein X) to the affinity ligand.
3)Wash away any unwanted stuff -> purified recombinant protein.
4)Investigate what can bind to Protein X.
pulldown assay: make a cell lysate
-Bacterial cell lysate doesn’t just contain the recombinant protein
-All soluble cellular proteins are there too
pulldown assay: Bind GST (+ Protein X) to the affinity ligand
-Only fusion protein will bind affinity ligand. And MFP, because they’re the same polypeptide
-Affinity ligand is usually attached to beads so it can be separated from other soluble stuff
pulldown assay: Wash away any unwanted stuff -> purified recombinant protein
-Other (unwanted) stuff has been washed away -> purified MFP (on a substrate that can be separated from other stuff)
pulldown assay: Investigate what can bind to Protein X.
-Now we can add stuff that we want to investigate:
=Cell lysate
=Another purified protein
=RNA / DNA / other molecules
-Remove Fusion protein form ligand – usually by competition with an excess of the free ligand
what are the steps in immunoprecipitation
-Almost identical technique and method to a pulldown.
-Uses antibodies to directly bind Protein X.
-See what interacts with it (co-immunoprecipitation).
advantages and disadvantages of immunoprecipitation
-Advantages
=Native protein can give better results (less background, fewer false positive)
-Disadvantages
=Antibodies are not easy to make: relatively expensive, not readily available, and very variable! Often limited in supply too.
=Need to recognise native protein
whats yeast two-hybrid
-Fusion proteins again
-Transcription factors are modular – separate domains for DNA-binding and transcription activation:
=Separate -> no transcription activation
=Bring together ->transcription activation
-DNA Binding Domain fused to a “bait”
-Transcription Activation domain fused to a “prey”
-If bait and prey interact, get transcription of the reporter gene…
whats ChIP
-Chromatin Immunoprecipitation
-Used to study interaction of proteins with DNA
-Use an antibody to purify your protein of interest (could also use a fusion protein too)
-Assay which DNA molecules are associated with your protein
-Have to cross-link DNA-protein first (or the interaction will not survive harsh purification conditions)
-DNA is one big molecule! Need to break it into bits (interested in local interactions)
-IP – anything not bound to antibody remains in solution (can also do a GST pulldown)
-DNA analysis – qPCR; microarray; ChIP-seq (uses Next Generation Sequencing to give a genome-wide view of the DNA sequences)
