L15 - Analysis of gene expression Flashcards
RNA expression and localisation - PCR based methods - Hybridisation-based methods. What are the expression and localisation types?
RNA expression:
Northern Blot
Microarray
RNA localisation:
Fluorescence In situ hybridisation
What is Northen blot - RNA expression?
- RNA is separated by electrophoresis
- RNA is transferred to a membrane to allow for blotting
- A gene-specific probe is added and hybridises to the target sequence
What are microarrays - RNA expression?
What would the results be? (–>)
Oligonucleotides are attached to a spot on a chip
Each spot has a different oligonucleotide, corresponding to a specific gene
RNA is prepared from a source and fluorescently-labelled cDNA is made from the RNA
Fluorescent cDNA is applied to the chip and allowed to hybridise
Lot of mRNA —>lot of cDNA —> lot of fluorescence
Not much mRNA —>not much cDNA —> low fluorescence
Black: No cDNA from either source
Yellow: Equal cDNA from both sources
Green: More cDNA from tumour cells
Red: more cDNA from normal cells
In RNA localisation what is, Fluorescent in situ hybridisation? (FISH)
Probe is often labelled with a fluorescent marker and visualised using microscopy
Can reveal RNA localisation within a chromosome, a cell, an organ, the whole organism
Correct RNA localisation can be vital for correct development.
In protein expression and localisation - what is western blotting?
- Proteins are separated by electrophoresis (based on size)…
- …then transferred to a membrane
(Similar principles to Northern blot)
- Detection is using a protein-specific antibody and a labelled secondary antibody
What is immunofluorescence?
The use of antibodies chemically labeled with fluorescent dyes to visualize molecules under a light microscope.
Not live imaging – a snapshot in time (cells usually need to be fixed and prepared in quite harsh ways)
Secondary antibody is usually conjugated to a fluorescent molecule – imaged using microscopy
Use different fluorophores – can see more than one molecule at a time
What are fusion proteins for protein localisation?
Fusion proteins are easy to visualise, they fuse the protein-coding regions together - in order to visualise the reporter in order to assess localisation.
What are fluorescent reporter genes?
Reporter genes are genes that enable the detection or measurement of gene expression. Could measure amounts of protein.
Reporter genes:
Easy to visualise (like GFP)
Or easy to assay (luciferase and β-galactosidase)
Fusion proteins are a type of reporter genes.
What are protein-protein interactions? and what are protein-DNA interactions?
Protein-protein interactions:
Pull-down assay
Yeast two-hybrid
Protein-DNA interactions:
Chromatin Immunoprecipitation
What is pull-down assay?
A way of analysing protein interactions in vitro: It uses a fusion protein that has an affinity for a specific ligand.
Fuse to the protein-coding sequence of your favourite gene and immobilise ligand on a surface. The fusion protein will bind & bring your fav protein.
What is Yeast two-hybrid?
Fusion proteins.
The “bait” is fused to a DNA-binding domain
The “prey” is fused to a transcription activation domain
Both fusion products are expressed inside a yeast cell, along with…
A reporter gene with a promoter that can be bound by the DNA-binding domain
If bait and prey interact, this will bring the transcription activation domain to the promoter and induce expression of the reporter gene
Report gene is often β-galactosidase – easy to assay as it produces a blue colour
What is Chromatin Immunoprecipitation (ChIP)?
Used to study interaction of proteins with DNA in a living cell.
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
