Analysis of gene expression Flashcards
Semester 1 year 1
What 3 methods can be used to analyse RNA expression and localisation?
-PCR based methods
-Hybridisation based methods
-Reporter genes
What are 2 types of PCR-based methods?
-reverse transcriptase PCR- quantitative or non-quantitative
-quantitative PCR
What are 3 types of hybridisation-based methods?
-Northern Blot
-microarrays
-fluorescence in situ hybridisation
What are hybridisation-based methods based on?
-properties of DNA/RNA
-complementary sequences will hybridise to each other
What is a probe?
DNA sequence that’s complementary to the RNA sequence that you want to analyse + has a label attached
Of the hybridisation-based methods, which determine RNA expression and which determine RNA localisation?
-RNA expression - Northern Blot + microarray
-RNA localisation - fluorescence In situ hybridisation
Describe the Northern Blot
-RNA separated by electrophoresis
-RNA transferred to a membrane to allow for blotting
-gene-specific probe is added + hybridises to the target sequence
Describe microarrays
-oligonucleotides are attached to a spot on a chip
-each spot has a different oligonucleotide corresponding to a specific gene
-RNA prepared from a source + fluorescently labelled cDNA is made from RNA
-fluorescent cDNA applied to chip + allowed to hybridise
What are oligonucleotides?
Short DNA molecules
What do microarrays measure?
Relative mRNA levels
Describe fluorescence In situ hybridisation
-same principle as Northern Blot
-probe labelled with fluorescent marker + visualised using microscopy
-correct RNA localisation can be vital for correct development
-can also be used to localise DNA
What colours are used to compare transcription levels?
-black - no cDNA from either source
-yellow - equal cDNA from both sources
-green - more cDNA from tumour cells
-red - more cDNA from normal cells
Describe reporter genes
-they’re easy to visualise (fluorescent) or assay (enzyme)
-common enzymatic reporter genes - luciferase + beta-galactosidase
What can reporter genes be used for?
-protein localisation
-to measure expression (RNA level)
What are 2 methods to determine protein expression + localisation?
-protein-specific antibodies
-fusion proteins + reporter genes
Describe how to determine protein expression + localisation using protein-specific antibodies
-protein specific antibody (primary antibody) recognises antigens
-secondary antibody recognises + binds to primary antibody
-secondary antibodies are conjugated to a molecule that allows for detection
Describe Western blotting
-same as northern blot
-last step - recognition using primary + secondary antibodies
-usually conjugated to enzyme that produces light, which is detected
Describe how to determine protein expression + localisation using fusion proteins + reporter genes
-use molecular cloning for live imaging of protein localisation
-fuse the CoDing sequence (CDS) for protein of interest to that of a green fluorescent protein (GFP)
-fluorescent protein can be directly visualised
What 3 techniques are used to analyse protein-protein molecular interactions?
-pull down assay
-immunoprecipitation
-yeast two-hybrid
Describe how to analyse protein-protein interactions using a pull-down assay
-make a recombinant DNA molecule
-Break the cell open
-Bind GST (fusion protein) + protein X to the affinity ligand
-wash away unwanted stuff
-investigate what can bind to protein X
Describe how to analyse protein-protein interactions using immunoprecipitation
-same as pull-down assay
-uses antibodies to directly bind to protein X
-see what interacts with it
Describe how to analyse protein-protein interactions using yeast two-hybrid
-transcription factors have separate domains for DNA binding + transcription activation
-separate = no transcription, together = transcription
-fuse DNA binding domain to a ‘bait’
-fuse transcription activation domain to a ‘prey’
-if bait + prey interact, get transcription
Describe how to analyse protein-DNA molecular interactions
-chromatin immunoprecipitation (ChIP)
-use antibody to purify protein
-crosslink DNA to protein
-DNA has to broken into bits as it’s very long
-immunoprecipitation - anything not bound to antibody remains in solution
-DNA purification + analysis
