Hybridisation techniques and RNA-seq

Question 1

Define nucleic acid hybridisation.

Answer 1

The forming of a double-stranded molecule between a single-stranded DNA/RNA and a single-stranded DNA probe.

Question 2

Does Southern blotting detect DNA or RNA sequences?

Answer 2

DNA.

Question 3

Does Southern or Northern blotting involve RNA-DNA hybridisation?

Answer 3

Northern.

Question 4

Before DNA is transferred to the membrane in Southern blotting, what technique is used?

Answer 4

Gel electrophoresis.

Question 5

Autoradiography is the final step of Southern blotting. True or false?

Answer 5

True.

Question 6

Northern blotting detects specific RNA sequences. True or false?

Answer 6

True.

Question 7

What are two key differences between the Southern and Northern blotting processes that are attributable to RNA as a molecule?

Answer 7

1. No digestion needed in Northern blotting (RNA is shorter).
2. No denaturation needed in Northern blotting (RNA is ss).

Question 8

Southern blotting detects gene expression. True or false?

Answer 8

False.

Question 9

What are three limitations of Northern blotting?

Answer 9

1. Limited number of target RNA sequences per membrane.
2. Requires a large amount of pure RNA.
3. Low sensitivity.

Question 10

In cDNA synthesis, first-strand synthesis uses mRNA as a template. True or false?

Answer 10

True.

Question 11

What reverse transcription primers anneal to the poly(A) tail in first-strand synthesis?

Answer 11

oligo(dT) primers.

Question 12

What enzyme is required for cDNA second-strand synthesis?

Answer 12

DNA polymerase.

Question 13

The template for second-strand cDNA synthesis is mRNA. True or false?

Answer 13

False.

Question 14

Glass chips that arrange thousands of DNA probes on a grid are called…

Answer 14

Gene expression microarrays.

Question 15

In microarrays, the sample is added to the probes. True or false?

Answer 15

True.

Question 16

In microarrays, are the sample or the probes labelled?

Answer 16

The sample is labelled.

Question 17

What technique compares RNA abundance between two samples?

Answer 17

Two-colour microarrays.

Question 18

Do two-colour microarrays use cDNA or mRNA?

Answer 18

cDNA.

Question 19

What are two limitations of microarrays?

Answer 19

1. Relative - based on comparison.
2. Requires large amounts of pure RNA.

Question 20

What technique can analyse the quantity and the sequence of RNA samples?

Answer 20

RNA-seq.

Question 21

RNA-seq does not use cDNA. True or false?

Answer 21

False.

Question 22

RNA-seq utilises next-generation sequencing. True or false?

Answer 22

True.

Question 23

What does it mean to ‘selectively enrich’ the RNA prior to RNA-seq?

Answer 23

To filter out target mRNAs from the total RNA of the sample.

Question 24

What part of mRNA transcripts is key to selective enrichment?

Answer 24

Poly(A) tails.

Question 25

What are two advantages of RNA-seq?

Answer 25

1. No prior knowledge of sequences needed.
2. Non-relative measurement.

Question 26

Southern blotting involves DNA-DNA hybridisation. True or false?

Answer 26

True.

Question 27

Which microarray looks at both intensity and colour?

Answer 27

Two-colour microarrays.