RNA

Question 1

What’s hnRNA? What sets it apart from other RNAs?

Answer 1

Pre-mRNA.
- no 5’cap
- no 3’polyA
- includes introns

Question 2

does RNAse A cleave ssRNA or dsRNA?

Answer 2

ssRNA

Question 3

What does RNAse H cleave?

Answer 3

DNA/RNA hybrids.

Question 4

What 3 RNA properties are taken into account when quantifying the quality of RNA?

Answer 4

1) Purity (260/280)
2) Integrity (gel / bioanalyser)
3) Quantity

Question 5

Explain northern blotting.

Answer 5

Both northern- and southern blots are based on probing RNA and DNA respectively with complementary sequence w/ binding site for radio-label.

1) Agarose gel electrophoresis.
2) Transfer from gel to membrane.
3) Immobilization of RNA via UV or heat.
4) Radio-label the membrane (binds to RNA).
5) Visualize RNA on x-ray table.

Question 6

Explain in situ hybridisation.

Answer 6

1. Cells are fixated.
2. Probes are deigned (nicks are induced, fluorescent dNTP is added to sequence –> probe).
3. Hybridisation in cells.
4. Wash away unbound probes.
5. Detect.

The difference from immunohistochemistry is the fluorescent component? FISH: Probe, IHC: Antibodies.

Question 7

Explain what nanostrings are.

Answer 7

1. Design reporter- and capture RNA sequence which contains fluorescent probes.
2. The reporter- and capture RNA will hybridise with the RNA of interest, emitting a combination of fluorescent wavelengths
3. When reporter- and capture probe has bound the RNA target, the complex is immbolized by the capture probe binding to the surface of the well and an electric current beig added.
4. Fluorescence detectors pick up the different “bar codes” of fluorescence, counts each barcodes and gives an idea of transcript abundance.

Question 8

What can a luciferase assay detect?

Answer 8

Transcription rate! Not transcription amounts.¨

Luciferase is conjugated to a certain promoter in a plasmid. Depending on if the promoter gets activated or not, the luciferase will be produced. If produced, in the presence of its substrate, light will be emitted. You can assay promoter strenght and/or the effect of cellular conditions of promoter activity.