Lec 12: Technologies in RNA and Protein Analysis

Question 1

Gene expression as measured by RNA

Answer 1

You can have transcription without subsequent translation

Question 2

What are some experimental methods for analysis of RNA levels?

Answer 2

Northern Blots
RT-PCR
RNA-seq

Question 3

Describe a Northern Blot

Answer 3

it analyzes mRNA levels

• The cells are manipulated in some way, the RNA is extracted, and run through a gel.
  -Then the electricity is run perpendicular, which pulls the RNA onto a blotting paper.
• A probe is washed over, which hybridizes to a single RNA transcript, and has some type of marker on it.

The mRNA amount can be quantified with brightness level, and there is a ladder to see the size of the fragment.

Question 4

What is RT-PCR?

Answer 4

RT: reverse transcription, turns it into cDNA, because PCR only works on DNA.

can observe very small amounts of mRNA and low levels of expression.

But because the non-linearity of PCR expansion (copies exponentially), it’s hard to quantify starting levels. (ie what does 2x brighter mean after 30 cycles?)

Have to design multiple custom primers for each gene

Question 5

What are some options for looking at a RT PCR?

Answer 5

1) only look at the one gene you are interested in
2) look at all the genes, not not just one at a time, and get a complete picture. (transcriptomics)

Question 6

How to real-time measurements work during a PCR?

Answer 6

Add a chemical that binds to a double-stranded DNA and fluoresces. Measure the intensity as a function of the number of cycles run. Once the fluorescence reaches a certain threshold, there is a formula for what that means in terms of the initial amount.

Question 7

Transcriptomics

Answer 7

Question 8

How do you study gene function?

Answer 8

Figure out what happens if the gene is perturbed (not expressed)

Translation is easy to mess up…you just need a small deletion at the right spot

To stop transcription is much harder.

Question 9

How do you get rid of a transcription?

Answer 9

RNAi: lets you knockout a gene without changing the genome. Does not stop transcription, just stops the amount of RNA by getting rid of it once it’s made (either by actively degrading it, or by blocking translation by binding and sitting there)

Question 10

How does RNAi compare to other therapeutic agents?

Answer 10

There is a lot of hype, but the difficulty is getting it into the cell

Question 11

How are protein levels detected?

Answer 11

It is harder–there’s no complementary sequence, so it needs to rely on antibodies that are unique to each protein, and each modification of that protein (ie phosphorylation)

Question 12

What is epitope tagging?

Answer 12

Inserting a sequence with a known antibody that binds to it somewhere in the sequence of the protein of interest.

Question 13

How is gel electrophoresis used for protein detection?

Answer 13

Size and charge matters

The protein needs to be denatured so you’re really only sorting by size, not by other attributes

Question 14

Western Blot

Answer 14

detects proteins (different from the Northern blot, that detects RNA

lysing the cells gets all the proteins, so need a antibody with a fluorescent tag or something like that.

Question 15

How can we determine what proteins bind to each other?

Answer 15

This only works in yeast, not bacteria.

Only works for proteins in the nucleus

Question 16

How can we see what proteins bind to each other when they are not in the nucleus?

Answer 16

Question 17

What does each of the following measure?
RT-PCR
Western Blot
Northern Blot
FRET
RNA-seq
microarray

Answer 17

RT-PCR: RNA
Western Blot: protein
Northern Blot: RNA
FRET: protein
RNA-seq: RNA
microarray: RNA