Studying gene expression II

Question 1

process of northern blotting

Answer 1

1. RNA separated by agarose gel electrophoresis
2. transferred onto nitrocellulose membrane
3. blotting with radioactive DNA probe specific to gene(s) of interest
4. autoradiography

Question 2

S1 nuclease protection assay

Answer 2

• RNA hybridised to raidolabeled, ss DNA (target specific)
• digestion with S1 nuclease (cleaves only ss DNA)
• gel electrophoresis + autoradiography

Question 3

real time qPCR

Answer 3

RNA > first strand cDNA > PCR+fluorescent dye

Question 4

qPCR variants

Answer 4

• CYBR-Green
• Taqman
• droplet digital PCR (DD-PCR)

Question 5

In situ hybridisation

Answer 5

• detection of transcripts in tissue sections

Question 6

probe used in insitu hybridisation

Answer 6

complementary DNA or RNA

Question 7

detection methods in in-situ hybrisation

Answer 7

• autoradiography
• fluorescence microscopy
• immunohistochemistry

Question 8

green fluorescent protein (GFP)

Answer 8

protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range

Question 9

reporter gene assays

Answer 9

reporter gene is fused with gene of interest; its activity reflects the activity of the regulatory elements controlling the expression of the gene of interest

Question 10

dual-glo luciferase assays

Answer 10

activity of the primary reporter is correlated with the effect of specific stimuli.
activity of the co-transfected control report = internale control to normalise results

Question 11

reporter gene assays; NanoLuc

Answer 11

• derived from shrimp luciferase
• smaller
• 100x brighter than firefly or renilla luciferase
• useful for fusion proteins