Lecture 6

Question 1

Name the types of expression profiling

Answer 1

Northern blot, arrays, semi-quantitative RT-PCR, RT-PCR, RNA sequencing

Question 2

What is the process of northern blotting with RNA and southern blotting with DNA

Answer 2

1) RNA/DNA run on agarose gel
2) Buffer blots nucleic acid onto filter paper via capillary action
3) Fix nucleic acid onto the filter paper
4) Probe filter paper with labelled probes of desired sequence
5) Wash away any unbound probe, expose to X-ray film to visualise
6) Develop an autoradiogram

Question 3

What are the steps of semiquantitative RT-PCR

Answer 3

1) Isolate total cell RNA
2) Synthesise cDNA using poly dT primer for first strand primer
3) Preform PCR using target sequence primers.

Question 4

What is the method of microarraying

Answer 4

1) Load pipetting tool robot with sequence
2) Reference and experimental sample and extract mRNA
3) cDNA synthesis by reverse transcriptase
4) Label with fluorescent dyes
5) Wash over the surface of microarrays

Question 5

What is cluster analysis

Answer 5

grouping genes with similar expression profiles and potential shared functionality. Consistent patterns can also be used as a diagnostic signature.

Question 6

How does RNA sequencing work

Answer 6

Fragment with DNase or sonication ligate adapters to ends. Number of sequences ‘hits’ is directionally proportional to expression level.