Techniques 4: analysis of gene expression

Question 1

Describe the PCR-based method of DNA/RNA analysis

Answer 1

RT-PCR (quantitative or non-quantitative)
Can use quantitative to measure RNA levels
Can use non-quantitative to look at diff isoforms of RNA

Question 2

Describe the Northern Blot

Answer 2

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

Question 3

What are the cons of the Northern Blot?

Answer 3

Slow
Only telling us about 1 RNA at a time

Question 4

Describe the Mircoarray

Answer 4

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

Question 5

What 2 techniques can we use to analyse RNA expression?

Answer 5

Northern Blot

Microarray

Question 6

What are the pros/cons of microarray?

Answer 6

• 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

Question 7

What type of analysis can we do to look at RNA localisation?

Answer 7

Fluorescence in situ hybridisation (FISH)

Question 8

Describe FISH

Answer 8

• 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

Question 9

What is a primary antibody?

Answer 9

protein specific antibody

Question 10

What is a secondary antibody?

Answer 10

recognises the primary antibody

Question 11

What is a conjugate?

Answer 11

A modified antibody that has a detectable signal e.g fluorescence

Question 12

Describe Western blotting

Answer 12

1. Proteins are separated by electrophoresis (based on size)
2. Then proteins are transferred to a membrane
3. Detection using a protein-specific antibody and a labelled secondary antibody

Question 13

Describe immunofluorescence

Answer 13

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)

Question 14

Describe fusion proteins

Answer 14

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)

Question 15

Describe reporter genes. Give an example

Answer 15

→ 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

Question 16

Name 3 methods used to analyse protein-protein interactions

Answer 16

Pull-down assay
Immunoprecipitation
Yeast two-hybrid

Question 17

What is a method used to analyse Protein-DNA interactions?

Answer 17

Chromatin Immunoprecipitation

Question 18

Describe the molecule the pull-down assay uses

Answer 18

→ 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

Question 19

Describe the process of the pull-down assay

Answer 19

1. Make a recombinant DNA molecule
2. Put recombinant DNA mol into E.coli = makes a recombinant protein
3. Protein is one polypeptide, has 2 domains (GST and your protein)
4. Make the cell lysate
5. Bind GAT (+ your protein) to the affinity ligand
6. Wash away any unwanted stuff = just purified recombinant protein left
7. Investigate what can bind to your protein

Question 20

Describe immunoprecititation

Answer 20

Almost identical method to pull-down assay
Uses antibodies to directly bind to protein
See what interacts with it (co-immunoprecipitation)

Question 21

Describe the process of yeast two-hybrid

Answer 21

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

Question 22

What is Chromatin Immunoprecipitation (ChIP) used for?

Answer 22

→ used to study interaction of proteins with DNA in a living cell
- uses antibody / fusion protein to purify protein of interest

Question 23

Describe the process of ChIP

Answer 23

1. Cross link DNA to your protein (bc if you try to purify without doing this, the DNA will not stay linked to your protein)
2. Chromatin Fragmentation - chop DNA up into fragments (few 100bp each)
3. Immunoprecipitation OR pull-down if used a fusion protein
4. DNA purification
5. DNA analysis:
   - PCR if you want to analyse a specific section
   - Whole genome