Studying gene expression

Question 1

What are the 2 approaches to studying gene expression?

Answer 1

• Look at the mRNA

- Look for protein

Question 2

What does a Northern blot look for?

Answer 2

Global RNA expression within a tissue

Question 3

Describe the process of carrying out a Northern blot

Answer 3

1. Separate RNA samples by electrophoresis
2. Place gel on top of wick paper and support in a tray of buffer solution
3. Place nitrocellulose filter (binds nucleic acids), blotting paper and weight on top of gel
4. Add radioactively labelled single stranded DNA
5. Study the results on an autoradiograph

Question 4

What is RT-PCR?

Answer 4

Reverse transcriptase PCR

Create cDNA to be used as a template to sequence genes of interest

Question 5

Describe the process of PCR

Answer 5

1. Prepare RNA from cells/tissue
2. Denature RNA at 90 degrees
3. Anneal short primers at 50 degrees
4. Heat to 70 degrees to activate reverse transcriptase enzyme so that primers are extended to form cDNA

Question 6

What is in situ hybridisation?

Answer 6

Detecting RNA according to its sequence in cells, tissues or embreyos

Question 7

How can proteins be used to investigate gene activity?

Answer 7

If the protein made by a gene has a known function, this can be measured experimentally

Question 8

When is a western blot used?

Answer 8

When you’re interested in where a gene is expressed for a protein

Question 9

Describe the process of a western blot

Answer 9

1. Blot a protein or within a cell/tissue
2. Raise a primary antibody against the protein in order to illicit an immune response
3. Raise a secondary antibody against the primary antibody which is conjugated with an enzyme (peroxidase or alkaline phosphatase) or a fluorescent chemical
4. Detect with a chemical reagent and microscope for fluorescence