Hybridisation techniques and RNA-seq Flashcards
Define nucleic acid hybridisation.
The forming of a double-stranded molecule between a single-stranded DNA/RNA and a single-stranded DNA probe.
Does Southern blotting detect DNA or RNA sequences?
DNA.
Does Southern or Northern blotting involve RNA-DNA hybridisation?
Northern.
Before DNA is transferred to the membrane in Southern blotting, what technique is used?
Gel electrophoresis.
Autoradiography is the final step of Southern blotting. True or false?
True.
Northern blotting detects specific RNA sequences. True or false?
True.
What are two key differences between the Southern and Northern blotting processes that are attributable to RNA as a molecule?
- No digestion needed in Northern blotting (RNA is shorter).
- No denaturation needed in Northern blotting (RNA is ss).
Southern blotting detects gene expression. True or false?
False.
What are three limitations of Northern blotting?
- Limited number of target RNA sequences per membrane.
- Requires a large amount of pure RNA.
- Low sensitivity.
In cDNA synthesis, first-strand synthesis uses mRNA as a template. True or false?
True.
What reverse transcription primers anneal to the poly(A) tail in first-strand synthesis?
oligo(dT) primers.
What enzyme is required for cDNA second-strand synthesis?
DNA polymerase.
The template for second-strand cDNA synthesis is mRNA. True or false?
False.
Glass chips that arrange thousands of DNA probes on a grid are called…
Gene expression microarrays.
In microarrays, the sample is added to the probes. True or false?
True.
In microarrays, are the sample or the probes labelled?
The sample is labelled.
What technique compares RNA abundance between two samples?
Two-colour microarrays.
Do two-colour microarrays use cDNA or mRNA?
cDNA.
What are two limitations of microarrays?
- Relative - based on comparison.
- Requires large amounts of pure RNA.
What technique can analyse the quantity and the sequence of RNA samples?
RNA-seq.
RNA-seq does not use cDNA. True or false?
False.
RNA-seq utilises next-generation sequencing. True or false?
True.
What does it mean to ‘selectively enrich’ the RNA prior to RNA-seq?
To filter out target mRNAs from the total RNA of the sample.
What part of mRNA transcripts is key to selective enrichment?
Poly(A) tails.
