27th Jan - Defining the Transcriptome Flashcards
How many protein coding RNAs are there in a human cell?
19553
What are retained introns?
Non-coding artefacts that weren’t spliced out
What are the two main forms of active genes based on their function?
House-keeping genes
Tissue specific genes
How do you identify which genes are housekeeping genes and which are tissue specific?
Compare the transcriptome of several different cell types. The overlapping region indicates the house-keeping genes
What percentage of protein coding genes are active in any given cell?
about 30%
How can we identify which genes are active?
Observe mRNA expression through RNA seq, RT PCR, Microarray, qRT PCR or Northern Blot
Look for protein expression e.g. Western Blot
Why is mRNA usually used to monitor gene expression?
It can be probed with high specificity due to base pairing of nucleic acids
Many transcripts can be probed simultaneously from the same sample
What is northern blotting?
An old technique to identify the expression of mRNA using a gel and labelled probes
Outline the process of northern blotting
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
When was Northern Blotting developed by Aluine, Kemp and Stark?
1977
What are the advantages of using Northern Blotting?
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
What are the disadvantages of Northern Blotting?
Labour intensive
Low throughput
What is RT PCR?
A process in which the RNA is amplified as cDNA
What is the use of RT PCR?
To test for gene expression, particularly in genetic diseases
Outline the process of RT PCR
Isolate RNA –> Heat RNA –> RT to make cDNA –> PCR amplify 1st cDNA stands using Taq polymerase –> Analyze products and run on gel
What is qRT PCR?
Measures flourescence intensity every cycle to monitor levels of mRNA expression in real time
How is the fluorescence visualised in qRT PCR?
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
What are microarrays?
A set of DNA sequences representing the entire set of genes of an organism, arranged in a grid pattern for use in genetic testing.
Outline a 1st generation microarray experiment
- Treated and control cells are labelled different colours
- Labelled cDNA from control and test samples are mixed
- Mixed labelled cDNA is hybridised onto the microarray and scanned
- The colour ratio indicates which cell expressed more of the gene
What is the key advantage of RNA sequencing?
It is not limited to existing sequence knowledge
What are the key challenges of RNA sequencing?
Establishing a cDNA library
Difficult to process such large quantities of data bioinformatically
Outline the process of basic RNA sequencing
mRNA –> RNA fragments or cDNA –> EST library w/adaptors –> Short sequence reads –> Mapped sequence reads
Outline the Illumina process of high throughput sequencing
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.
How was EZH2 identified as a therapeutic target in metastatic prostate cancer by Varambally (2002)?
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
What is the therapeutic target of GSK126 in Lymphoma?
EZH2
