27th Jan - Defining the Transcriptome

Question 1

How many protein coding RNAs are there in a human cell?

Answer 1

19553

Question 2

What are retained introns?

Answer 2

Non-coding artefacts that weren’t spliced out

Question 3

What are the two main forms of active genes based on their function?

Answer 3

House-keeping genes

Tissue specific genes

Question 4

How do you identify which genes are housekeeping genes and which are tissue specific?

Answer 4

Compare the transcriptome of several different cell types. The overlapping region indicates the house-keeping genes

Question 5

What percentage of protein coding genes are active in any given cell?

Answer 5

about 30%

Question 6

How can we identify which genes are active?

Answer 6

Observe mRNA expression through RNA seq, RT PCR, Microarray, qRT PCR or Northern Blot

Look for protein expression e.g. Western Blot

Question 7

Why is mRNA usually used to monitor gene expression?

Answer 7

It can be probed with high specificity due to base pairing of nucleic acids
Many transcripts can be probed simultaneously from the same sample

Question 8

What is northern blotting?

Answer 8

An old technique to identify the expression of mRNA using a gel and labelled probes

Question 9

Outline the process of northern blotting

Answer 9

Cell undergoes lysis and membrane disruption, ribonuclease activity is inhibited and the sample is deproteinized –> ssRNA –> Sample is run on a denaturing agarose gel (typically uses formaldehyde to denature) –> Transferred to a positively charged nylon membrane using UV or vacuum gel transfer –> Block using salmon sperm DNA –> Hybridize membrane w/ labelled probes –> Visualise on an X-ray film

Question 10

When was Northern Blotting developed by Aluine, Kemp and Stark?

Answer 10

1977

Question 11

What are the advantages of using Northern Blotting?

Answer 11

Definitive experiment to determine mRNA levels
Can measure the size of the transcript and compare it to the ORF
Can detect multiple mRNA species - alternative splicing
Relatively inexpensive

Question 12

What are the disadvantages of Northern Blotting?

Answer 12

Labour intensive

Low throughput

Question 13

What is RT PCR?

Answer 13

A process in which the RNA is amplified as cDNA

Question 14

What is the use of RT PCR?

Answer 14

To test for gene expression, particularly in genetic diseases

Question 15

Outline the process of RT PCR

Answer 15

Isolate RNA –> Heat RNA –> RT to make cDNA –> PCR amplify 1st cDNA stands using Taq polymerase –> Analyze products and run on gel

Question 16

What is qRT PCR?

Answer 16

Measures flourescence intensity every cycle to monitor levels of mRNA expression in real time

Question 17

How is the fluorescence visualised in qRT PCR?

Answer 17

2 main methods:
Taqman Probe - probe uses a reporter and quencher, when free in solution the quencher prevents signal, when it is attached to ss cDNA the nuclease activity of Taq polymerase –> quencher cleavage causing fluorescence. With each cycle of PCR more dye molecules are released resulting in an increase in fluorescence intensity proportional to the amount of amplicaon synthesised

SYBR green - Probe only flouresces with dsDNA so every new cycle of PCR –> more fluorescence

Question 18

What are microarrays?

Answer 18

A set of DNA sequences representing the entire set of genes of an organism, arranged in a grid pattern for use in genetic testing.

Question 19

Outline a 1st generation microarray experiment

Answer 19

1. Treated and control cells are labelled different colours
2. Labelled cDNA from control and test samples are mixed
3. Mixed labelled cDNA is hybridised onto the microarray and scanned
4. The colour ratio indicates which cell expressed more of the gene

Question 20

What is the key advantage of RNA sequencing?

Answer 20

It is not limited to existing sequence knowledge

Question 21

What are the key challenges of RNA sequencing?

Answer 21

Establishing a cDNA library

Difficult to process such large quantities of data bioinformatically

Question 22

Outline the process of basic RNA sequencing

Answer 22

mRNA –> RNA fragments or cDNA –> EST library w/adaptors –> Short sequence reads –> Mapped sequence reads

Question 23

Outline the Illumina process of high throughput sequencing

Answer 23

RNA –> DS DNA –> Fragmentation by enzyme or sonication –> end repair –> adapter ligation –> flow cell hybridisation –> bridge amplification to create a cluster of fragments with the same sequence–> reverse strand removal –> flourescently labeled NTs are passed by each cluster, a computer reads which NT was incorporated. Flourescent label is cleaved and repeat.

Question 24

How was EZH2 identified as a therapeutic target in metastatic prostate cancer by Varambally (2002)?

Answer 24

EZH2 (a component of the polycomb repressive complex) = a histone methyltransferase which trimethylates H3K27 turning genes off.

Significance analysis of microarrays (SAM) identified 55 genes that were significantly upregulated and 480 genes that were signficantly down regulated in metastatic prostate cancer compared to localised prostate cancer - Key gene was EZH2

Used RT PCR of 18 prostate samples and cell lines which showed high EZH2 levels

Analyzed tissue extracts by western blotting which showed that levels of the EZH2 protein increased in malignant prostate cancer

–> EZH2 could be used as a biomarker for prostate progression and a possible therapeutic target

Question 25

What is the therapeutic target of GSK126 in Lymphoma?

Answer 25

EZH2