Analysis of gene expression

Question 1

What 3 methods can be used to analyse RNA expression and localisation?

Answer 1

-PCR based methods
-Hybridisation based methods
-Reporter genes

Question 2

What are 2 types of PCR-based methods?

Answer 2

-reverse transcriptase PCR- quantitative or non-quantitative
-quantitative PCR

Question 3

What are 3 types of hybridisation-based methods?

Answer 3

-Northern Blot
-microarrays
-fluorescence in situ hybridisation

Question 4

What are hybridisation-based methods based on?

Answer 4

-properties of DNA/RNA
-complementary sequences will hybridise to each other

Question 5

What is a probe?

Answer 5

DNA sequence that’s complementary to the RNA sequence that you want to analyse + has a label attached

Question 6

Of the hybridisation-based methods, which determine RNA expression and which determine RNA localisation?

Answer 6

-RNA expression - Northern Blot + microarray
-RNA localisation - fluorescence In situ hybridisation

Question 7

Describe the Northern Blot

Answer 7

-RNA separated by electrophoresis
-RNA transferred to a membrane to allow for blotting
-gene-specific probe is added + hybridises to the target sequence

Question 8

Describe microarrays

Answer 8

-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

Question 9

What are oligonucleotides?

Answer 9

Short DNA molecules

Question 10

What do microarrays measure?

Answer 10

Relative mRNA levels

Question 11

Describe fluorescence In situ hybridisation

Answer 11

-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

Question 12

What colours are used to compare transcription levels?

Answer 12

-black - no cDNA from either source
-yellow - equal cDNA from both sources
-green - more cDNA from tumour cells
-red - more cDNA from normal cells

Question 13

Describe reporter genes

Answer 13

-they’re easy to visualise (fluorescent) or assay (enzyme)
-common enzymatic reporter genes - luciferase + beta-galactosidase

Question 14

What can reporter genes be used for?

Answer 14

-protein localisation
-to measure expression (RNA level)

Question 15

What are 2 methods to determine protein expression + localisation?

Answer 15

-protein-specific antibodies
-fusion proteins + reporter genes

Question 16

Describe how to determine protein expression + localisation using protein-specific antibodies

Answer 16

-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

Question 17

Describe Western blotting

Answer 17

-same as northern blot
-last step - recognition using primary + secondary antibodies
-usually conjugated to enzyme that produces light, which is detected

Question 18

Describe how to determine protein expression + localisation using fusion proteins + reporter genes

Answer 18

-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

Question 19

What 3 techniques are used to analyse protein-protein molecular interactions?

Answer 19

-pull down assay
-immunoprecipitation
-yeast two-hybrid

Question 20

Describe how to analyse protein-protein interactions using a pull-down assay

Answer 20

-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

Question 21

Describe how to analyse protein-protein interactions using immunoprecipitation

Answer 21

-same as pull-down assay
-uses antibodies to directly bind to protein X
-see what interacts with it

Question 22

Describe how to analyse protein-protein interactions using yeast two-hybrid

Answer 22

-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

Question 23

Describe how to analyse protein-DNA molecular interactions

Answer 23

-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