8) Measuring Eukaryotic Gene Expression

Question 1

What does measuring eukaryotic gene expression mean?

Answer 1

How do we know which genes are being highly or less highly expressed under any particular set of conditions

Question 2

What are some examples of why and genes might be expressed at different levels?

Answer 2

Photosynthesis: A gene that encodes a photosynthetic protein= Might only be expressed when there is light available for photosynthesis= Gene might produce more transcripts when in light than dark

Responding to hormones: Mammalian cell will respond to hormone by switching on expression of appropriate genes= Hormone will bind to a receptor on the surface of the cell that needs to respond to hormone= Produce a signal that results in production of a hormone specific transcription factor that switches on expression of the hormone-responsive gene

Question 3

Why measure transcript levels?

Answer 3

1) Detecting that an organism has responded to something, a way of seeing what that response is= Can be from developmental signal or external condition

2) Diagnosing effects of a genetic difference
Looking to see if a patient can express a particular gene needed for good health= could learn more about how the genetic defect in a mutant manifests itself
Genetic mutants= Associate gene with a particular function

3) Also predicting which proteins might be produced= Much cheaper and easier to measure than to measure protein levels

Question 4

What are the different ways of measuring transcript levels? (names only)

Answer 4

A) Hybridisation- northern blots
B) cDNA-based methods
1. qRT-PCR
2. Microarrays
3. Next generation sequencing

Question 5

How do you use northern blot to measure transcript levels?

Answer 5

1) Extract total RNA from cells, run on a gel
2) Make radioactively-labelled DNA probe with a sequence matching gene of interest. Can make using PCR, does not need to cover whole of mRNA but just the specific part of the sequence
3) Probe will bind to each copy of the transcript
4) Intensity of band indicates AMOUNT of transcript on X-ray= Dark= High level

Question 6

How does running gel work?

Answer 6

Transfer RNA from the gel onto a nitrocellulose filter by blotting with a weight

Once RNA has been transferred to filter, it is mixed with radioactive probe and allowed to hybrid

mRNA for every other gene that does not match the probe= will not bind

Can then wash away= Leaves probe that attached to mRNA molecules on filter

X-ray film is used to detect where the highest levels

Question 7

How does transferring the RNA from gel onto a nitrocellulose filter work?

Answer 7

Many RNAs in sample will be spread out by size on gel BUT cannot see most of them

Need to transfer all of the mRNA to sold filter= thin sheet of nitrocellulose

RNA can bind nitrocellulose strongly= Will not be detached

Transfer of RNA to filter happens by simple capillary action= Draws buffer through the stack and this draws the RNA onto filter

Filter: Incubated with a radioactive DNA probe for gene of interest

Question 8

What are the indirect methods of measurement?

Answer 8

cDNA-based methods
They involve conversion of mRNA into cDNA and subsequent amplification steps

Therefore: Not directly measuring mRNA

Question 9

What is the example of using cDNA based methods instead of northern blot?

Answer 9

• It is non-radioactive
• Can measure the transcripts from many different genes in one experiment
• Most use amplification methods so only need small amounts of material (but indirect)

BUT: Cannot PCR on RNA as DNA polymerases only work on DNA

So: Need to convert RNA to DNA in order to be able to include an amplification step

Question 10

What does reverse transcriptase do?

Answer 10

Enzyme that goes against the central dogma

RNA —> DNA

Comes from viruses that have an RNA genome and make DNA copies to incorporate into the host genome

Enzyme: Uses RNA as a template and makes complementary DNA (cDNA) from it= Single stranded DNA copy of ssRNA

RNA viruses= Have RNA genome and therefore if they want to insert their genome into mammalian host= Need to convert to DNA

Question 11

How does reverse transcriptase make cDNA from mRNA?

Answer 11

1) Need PRIMER= every mRNA will have a poly A tail= Primer is poly T, same for each mRNA
2) Primer binds to tail= Reverse transcriptase extends the nucleotide chain from the primer back towards the 5’ mRNA= makes copy
3) Result= Double helix of one mRNA and one cDNA strand
4) Degrade most of the RNA with RNAse= Do not need RNA
5) Result: to leave a few short pieces to use as primers for next step

6) Synthesises complementary DNA strand using DNA polymerase
Overall: Makes complementary DNA to the cDNA= dsDNA

Question 12

How do you use cDNA to measure gene expression?

Answer 12

If sample is from cell or tissue that is transcribing gene of interest to high level= Lots of mRNA

Result= Lead to formation of a lot of cDNA

BUT: if less highly expressed gene= less cDNA

THEREFORE: you can measure cDNA levels which will be proportional to mRNA levels

cDNA is a lot easier to handle than RNA as it doesn’t degrade so easily + CAN do PCR on cDNA

By making cDNA copies of mRNA= Can now amplify these by PCR= Use 3 different methods to measure relative levels of transcripts

Question 13

cDNA-based methods:

What is qRT-PCR?

Answer 13

Quantitative Real-time PCR

IF you have more cDNA from a gene that is transcribing to higher levels= More template for PCR= More PCR product after a given number of cycles

PCR= cDNA contains copy of EVERY transcript in same, USE primers specific to gene of interest

Measure the relative amount of PCR product between samples= Can see which had more and which had less

Question 14

What is the problem with making PCR quantitative?

Answer 14

1) Low amount of template cDNA gives little product

BUT: Had to tell difference between more templates and lots of templates= Need more qualitative method

Question 15

What are the different phases during a PCR reaction? Which bit do you measure?

Answer 15

BASELINE: PCR plateau, if you measure the amount of PCR during each cycle, most of the first few cycles the amount seems to follow a baseline level= Too little to detect so even doubling the product each time, still cannot detect

EXPONENTIAL PHASE: MEASURE DIS
The sample which had more cDNA copies of the transcript, Will take less cycles to reach the ‘threshold’ that you’ve set

PLATEAU: Eventually, enzymes and reagents become limiting= no new product is made with increasing cycles (around 35-40), the two samples will look very much alike as the slow one will catch up with faster one

Question 16

What is used to detect the PCR product during qRT-PCR?

Answer 16

No need for gel

ADD FLUORESCENCE dye= Binds to newly synthesised DNA PCR product

Fluorescence will get BRIGHTER as the amount of product increases

Amount of product can be monitored in REAL TIME

Advantage of method: Once you have cDNA samples, can test them again and again with different primer pairs for different genes= Can get a lot of information out of one experiment

Question 17

cDNA based method:

What are microarrays?

Answer 17

Microarray chip: All of the little squares contain many, many dots which each represents a gene in the genome being studied= Tens of thousands of separate dots

Each dot is a gene ‘probe’ (oligonucleotides) specific for one of the genes= can only bind to that specific transcript

Each dot: Detect a specific transcript and to measure how many copies of that particular transcript there are present in a sample

Question 18

What is a probe?

Answer 18

Collections of oligonucleotides= Bits of DNA sequence that match and recognise a particular transcript

Sometimes: Probes are made up of several matching oligonucleotide primers

NOT radioactive

Question 19

How can you use microarrays to measure levels of gene expression?

Answer 19

1) Extract mRNA from the samples that want to be compared
2) Label each sample with either a red or green fluorescent dye
3) Labelled cDNAs are mixed and allowed to hybridise to microarray chip
4) Each probe will either bind red or green copies of cDNA for particular gene
5) Red and green signals are scanned by a laser and recorded

Comparing ratio of red to green= Can see if the gene was switched on

Each spot represents one gene= The more cDNA copies are present which bind to probe= Stronger the intensity of the red or green colour

Question 20

cDNA based methods:

What is next generation sequencing?

Answer 20

Determines sequence of millions of DNA molecules

Converts mRNA —> cDNA

Sequences all the copies

Can be used to measure levels of mRNA= Convert RNA to cDNA then determine sequence of all copies of cDNA

Count how many times it has been sequenced= Relative abundance

If you sequence same bit of cDNA a lot of times= Lots of mRNA

IF only a few sequence reads for a piece of cDNA= Low levels of mRNA in samples

Question 21

What are the comparisons between microarrays and Next generation sequencing?

Answer 21

• Microarray requires a chip with a probe for every gene. -Need to know your genome sequence.
• Microarray data is fairly simple to analyse (ratio between red/green).
• Fairly expensive.

RNAseq:

• Less genome information required to do RNAseq.
• Good for less well studied genomes.
• RNAseq data is complicated to analyse.
• Fairly expensive.