Techniques 4: analysis of gene expression Flashcards
Describe the PCR-based method of DNA/RNA analysis
RT-PCR (quantitative or non-quantitative)
Can use quantitative to measure RNA levels
Can use non-quantitative to look at diff isoforms of RNA
Describe the Northern Blot
Measures RNA expression:
1. RNA separated by electrophoresis (small RNAs at bottom)
2. RNA is transferred to a membrane to allow for blotting
3. A gene-specific probe (labelled radioactive) is added and hybridised to the target sequence
What are the cons of the Northern Blot?
Slow
Only telling us about 1 RNA at a time
Describe the Mircoarray
Detects RNA expression:
1. Oligonucleotides attached to a spot on a chip
2. Each spot has a different oligonucleotide, corresponding to a specific gene
3. RNA prepared from a source and fluorescently labelled cDNA is made from the RNA
(cDNA acts as a proxy for RNA levels)
4. Fluorescent cDNA is applied to the ship and allowed to hybridise
Alot of mRNA → alot of cDNA → alot of fluorescence
What 2 techniques can we use to analyse RNA expression?
Northern Blot
Microarray
What are the pros/cons of microarray?
- 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
What type of analysis can we do to look at RNA localisation?
Fluorescence in situ hybridisation (FISH)
Describe FISH
- Principle of this is similar to Northern blot
- Probe is often labelled with a fluorescent marker and visualised using microscopy
- DNA/RNA localisation within a chromosome, a cell, an organ, the whole organism
- Correct RNA localisation can be vital for correct development
What is a primary antibody?
protein specific antibody
What is a secondary antibody?
recognises the primary antibody
What is a conjugate?
A modified antibody that has a detectable signal e.g fluorescence
Describe Western blotting
- Proteins are separated by electrophoresis (based on size)
- Then proteins are transferred to a membrane
- Detection using a protein-specific antibody and a labelled secondary antibody
Describe immunofluorescence
Secondary antibody usually conjugated to a fluorescent molecule - imaged using microscopy
Use different fluorophores
Not live imaging - a snapshot in time (cells usually need to be fixed and prepared harshly)
Describe fusion proteins
Fuse to a protein easy to visualise
Fuse the 2 protein coding sequences (CDS) - visualise the reporter to assess localisation
Fluorescent proteins are commonly used - visualised by microscopy
(other fusion proteins exist for different applications)
Describe reporter genes. Give an example
→ easy to visualise (like GFP)
→ or easy to assay (luciferase and B-galactosidase)
Common gene is LacZ = makes beta-galactosidase, which produces a blue precipitate
Name 3 methods used to analyse protein-protein interactions
Pull-down assay
Immunoprecipitation
Yeast two-hybrid
What is a method used to analyse Protein-DNA interactions?
Chromatin Immunoprecipitation
Describe the molecule the pull-down assay uses
→ 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) → like a bead
Describe the process of the pull-down assay
- Make a recombinant DNA molecule
- Put recombinant DNA mol into E.coli = makes a recombinant protein
- Protein is one polypeptide, has 2 domains (GST and your protein)
- Make the cell lysate
- Bind GAT (+ your protein) to the affinity ligand
- Wash away any unwanted stuff = just purified recombinant protein left
- Investigate what can bind to your protein
Describe immunoprecititation
Almost identical method to pull-down assay
Uses antibodies to directly bind to protein
See what interacts with it (co-immunoprecipitation)
Describe the process of yeast two-hybrid
Way of identifying things that you didn’t know interacted with your protein in the first place
1. DNA Binding Domain fused to a bait’
2. Transcription Activation domain fused to a ‘prey’ (something you think interacts with your protein of interest)
3. Put these into a yeast cell: If bait and prey interact, there will be transcription of the reporter gene
What is Chromatin Immunoprecipitation (ChIP) used for?
→ used to study interaction of proteins with DNA in a living cell
- uses antibody / fusion protein to purify protein of interest
Describe the process of ChIP
- Cross link DNA to your protein (bc if you try to purify without doing this, the DNA will not stay linked to your protein)
- Chromatin Fragmentation - chop DNA up into fragments (few 100bp each)
- Immunoprecipitation OR pull-down if used a fusion protein
- DNA purification
- DNA analysis:
- PCR if you want to analyse a specific section
- Whole genome
