Derek Huntley Flashcards
What is the aim of Lecture 1?
Learning how Gene Expression is measured and what techniques/technology has been developed to help us.
What does the word ‘genomics’ refer to?
Genomics is basically…
- Identifying location of genes
- Annotating them (including transcripts)
- Looking at how they are regulated
How Northern blot used to measure gene expression?
Simplest way to measure gene expression - Northern Blot
- Extract RNA
- Run Gel Electrophoresis - seperation by size
- Transfer RNA to a membrane
- Add a labelled probe that can hybridize to specific sequences
- Visualize using X-ray
The Degree of label (fluorescence or radioactivity) is correlated to the quantity present.
What trying to quantify Northern blot results - what should we remember to do?
Compare the target gene to internal control and adjust our result accordingly
Normalize our results by examining the change in fluorescence in target gene and our control
Generally speaking - accuracy is quite poor - example below we observe roughly 5 fold increase
Why do we do this?
Control for how much sample we start with and how much we end with.
For example, your experimental sample might just start with higher levels of mRNA to begin - by chance oberseve a significant increase in fluorescence
Briefly outline how the normal PCR procedure works.
- Denaturation
- Primer annealing - one on each strand in the 3’ region
- Elongation using taq polymerase
Note - if you were to amplify RNA then…
The number of copies after several rounds of replication are directly proportional to the initial mRNA level - hence, this can be taken advantage of in order to measure RNA levels
Outline the process of qPCR.
Describe whats happening the following qPCR graph.
How is qPCR normalisation performed?
Northern blot and qPCR limitations?
What are Microarrays?
Example of Microarray?
How do cDNA chips and Affy Array differ?
Note - Both use fluoresecent tags that bind to DNA allowing us to visualize the expression
How is Affy chip visualized/analysed?
How is cDNA chip visualized/analysed?
Microarray Limitations?
What is RNA-seq? What are its benefits?
General overview of RNA-seq process?
RNA-seq method - how can we can gauge the number of copies of a gene present?
What are the two ways used to normalize RNA-seq results?
What is RPKM/FPKM Normalisation?
Raw Read Count Normalisation - RNA-Seq Normalisation?
Remember this is RNA-Seq –> we obtain reads which map to a specific gene
- Take the mean of gene counts across all samples - Mean number of times a gene is expressed - expect it to be similar
- Divide gene count for a specific sample by the geometric mean –> use several
- Get a list of ratios - we take the median of this this list to be the normalization factor
Can RNA-seq be used to identify splice variants?
What is one of the main problems associated with RNA-seq - have created a solution for this?
Hint - Heterogeneity of the sample
What is the workflow (procedure) associated with Single Cell RNA-seq?
Single Cell RNA-seq Data Analysis performed?
Conclusions from gene expression, microarrays and RNA seq?
Conclusions
- Analysis of gene expression essential in bioscience research
- Microarrays revolutionised biological research enabling high throughput analysis –> They can be used to measure gene expression and also co-expression
But!
Ability to detect low expression levels not possible with microarrays, noisy, lacks recognition of transcript variantion/novel trasncripts, etc.
- Microarrays now superseded by RNA-seq –> RNA-seq provides far greater accuracy and can identify exact transcript –> Can also identify previously unknown transcripts
- Advancement has led to single cell RNA-seq –> Enables resolution of gene expression at the level of individual cells
- RNA seq used to provide candidate genes that expressed different to be further analyzed.
- Enables the identification of heterogeneity in cell populations
What does -seq mean?
Anything that has -seq at the end involves next gen sequencing
Lecture 2 aims?
DNA methylation review
How is DNA methylation performed in mammalian cells? WHat enzymes are involved?
How is methylation passed on during mitosis?
Is there a link between DNA methylation states and Disease?
In the genome, where Does DNA Methylation Occur?
Not referring to CpG islands!
What is the role of DNA methylation at intergenic regions?
Outline two examples whereby DNA methylation helps to maintain genomic integreity.
Aberrant - abnormal
What is the role of DNA Methylation around Repetitive Elements?
What is the link between DNA methylation and Cancer?
DNA Methylation Conclusions?
Outline how MeDIP-seq is used to identify DNA Methylation?
What is the following plot meant to depict?
Outline how Bisulphite (Bisulfite) Sequencing is performed?
Alternative to MeDIP-Seq
What does X-inactivation refer to?
What are Long Non-Coding RNA Sequences?
What is Xist? How does it allow for X chromosome inactivation?
HOTAIR is another example of a lncRNA, what does it do?
How do we identify what regions of DNA are associated with histone modifications?
What is the role of transcription factors?
Transcription factors either up- or down-regulate a gene
What are some examples of mechanisms used to regulate gene expression?
They use several mechanisms to regulate gene expression, including:
a) Stabilizing or blocking the binding of RNA polymerase to DNA
b) Recruit coactivator or corepressor proteins to the transcription factor DNA complex
c) Catalyse the acetylation or deacetylation of histone proteins
Reminder
- Histone acetyltransferase (HAT) activity – acetylates histone proteins. Weakens the association of DNA with histones making the DNA more accessible to transcription. Transcription is up-regulated
- Histone deacetylase (HDAC) activity – deacetylates histone proteins. Strengthens the association of DNA with histones making the DNA less accessible to transcription. Transcription is down-regulated
How do we identify TF binding sites?
What is ChIP-seq?
Outline the Chip-seq procedure.
Lecture 3 aims?
What is an SNP?
Why Are SNPs Important?
What are the 4 different possible locations of an SNP?
Relationship between SNPs and diseases?
What are coding SNPs?
These are SNPs located in the coding region of a gene
What are transitions and transversions? What is the Ti/Tv substitution ratio?
What is Sickle Cell Anaemia? Where is it commonly found? What are the common symptoms?
Cause of Sickle cell anaemia?
Outline how different SNP variants of ApoE may result in different probabilities of developing Alzheimer’s Disease.
Are non-coding SNPs associated with disease, if so what regions are normally impacted?
Why is indentifying non-coding regulatory SNPs difficult?
What are the two ways a SNP can disrupt a splice site?
Explain one of the two using - OAS1 Gene as an example
Is the disruption of splice sites commonly associated with disease?
Will SNPs in auxillary splice sequences impact splicing?
Outline how SNP results in the inlcusion of a cryptic splice site in the BRCA2 gene.
What are the three main effects of SNPs on splicing?
Have numerous effects:
- Delete splice site
- Change Splice site
- Introduce a cryptic exon
What are the 3 different impacts of a insertion/deletion of a base?
What is a genome-wide associations (GWAS) study?
What is the 100,000 Genomes Project?
How do we identify variants (SNPs) from a GWAS?
GWAS Challenges?
What is Linkage disequilibrium? Why is Linkage disequilibrium important to consider when performing a GWAS?
What is Expression Quantitative Trait Loci (eQTL)? Principle behind it?
Difference between a Trans and Cis eQTLs?
Are GWAS and eQTL normally performed together?
How is SNP Genotyping performed – How do we check whether an individual has these SNPs?
What does heteroplasmy refer to?
Are there disease associated with the mitochondria/heteroplasmy?
Lecture 3 - SNP overview
1. SNPs are an important area of genomic variation
- They can affect disease development, as well as response to pathogens, chemicals and drugs
- They are of major importance in biomedical research for aiding in localisation of disease causing genes, identifying disease causing genes and also possible likelihood of developing disease - genotyping
- SNPs can affect proteins directly by altering amino acids or protein structure
- They can also alter proteins indirectly by altering promoters and other transcription factor binding sites (TFBS)
2. The 100,000 genomes project aims to identify SNPs associated with disease using GWAS as a resource for treatment and research - GWAS is a rapidly developing and significant area of research
What is ENCODE?
ENCODE - Encyclopaedia of DNA Elements - identifies regulatory regions of genes - genome annotation
It is was used as an example of what you can do if you combine multiple techniques to identify and understand regulatory regions of genes
Lecture 4 - Learning objectives?
What is the ENCODE project?
What are regulatory regions very hard to identify?
In a nutshell what is the ENCODE Project?
What functional elements are we looking for in the human genome?
What are DNA Hypersensitive Sites (HS)? Why are we interested in them?
Outline how DNase-seq is used to identify DNA Hypersensitive Sites.
End-capture?
Double Hit?
What is DNase-seq Footprinting?
Basically…
Using DNase-seq to identify open regions that (non-compacted), sequencing it and mapping back to the genome to identify regulatory regions
What is DNA Foot printing (not DNAse-seq)?
What is FAIRE-Seq - Alternative to DNA-seq?
Difference between FAIRE-Seq and DNase-Seq?
What is ATAC-seq?
Summary of regulatory site identification methods
Note….
- MNase–seq uses Micrococcal nuclease
- All doing the same thing just fragmenting and isolating the DNA using different techniques
- ChiP-seq is isolating DNA with TF bound but the others are identifying open DNA
Why do we care about Chromatin Interaction? What do we use to study it?
Go step by step through the traditional ChIA-PET protocol.
What does the advanced/improved ChIA-PET involve?
General Principle behind ChIA-PET?
- Isolating chromatin complex
- Identifying DNA ends that associate with this complex by ligating them together (stitching two different DNA regions together)
- Sequencing these regions to know what regions of DNA are interacting at this chromatin complex
How is ChIA-PET Interpreted?
What were the two main DNA methylation site detection techniques previously discussed?
Solution to the expensive nature of WGBS?
When performing size selection in RRBS, what sizes do we select for?
The benefit of this method is that the methylation areas of interest are clustered, CpG islands and promoters.
The fragments are size filtered at about 40-220 bases, which represents the majority if these sequences (CpG islands and promoters)
This avoids the disparate methylation sites, which generally are not of interest. The method does lose some information but focusses on the main sites of methylation.
Outline the different splicesome players and auxillary sequences involved in splicing?
What is RIP-seq, what is it used for?
RIP-seq – identify RNA-protein interaction – useful for understanding splicing
- Methods similar to DNA-protein interaction identification - Extract RNA with protein, add Antibody – immunoprecipitation, purify RNA, sequence
- RIP-seq involves immunoprecipitation (IP) of RNA-binding protein (RBP) of interest
- RNA bound to the RBP can be isolated for sequencing in this IP provided that the isolation is done non-stringently
- Consequence - this low stringency results in low specificity - We don’t know specifically where the protein binds on the RNA
What technique with a higher specificity can be used in place of RIP-seq?
Outline the CLIP-seq procedure.
Variants of CLIP-seq technique?
What do -seq methods have in common?
What is the controversy behind ENCODE?
Lecture 4 conclusions?
Lecture 5 objectives?
What is epigenetics?
Are epigenetics and cancer interlinked?
What is a classic example of epigenetics?
How can X inactivation manifest itself - Example?
How does X-inactivation manifest itself in Cats?
Given that these mice are genetically identical, what could possibly explaining the observed variation?
Explain how epigenetic modification of the agouti gene leads to two completely different offspring.
Link between epigenetics and health (Cancer, aging & other diseases)
What people are ideal to study epiegentic changes?
What historical evidence is there for epigenetic modifications being inherited?
Evidence - Dutch Famine
What are the three possible modes of epigenetic inheritance?
Why is there difficulty in identifying Epigenetic Inheritance?
What happens to DNA methylation states, are they earsed or passed on?
What happens to most methylated DNA? How does some methylation get passed on?
What are imprinted genes?
How does imprinting work?
Why do our germline cells need to reset/erase methylation on all imprinted genes?
We need to wipe clean the methylation as the gametes develop because at the beginning of meiosis the cell will have both Male and Female imprinting patterns –> Sperm needs to have the male methylation pattern thus we need remove the female methylation pattern which is provided by the egg upon fertilization.
Genomic imprinting example - IGF2
Outlien another example of imprinted gene - IGF Receptor (IGFR2).
Outline another example of imprinted genes - DLK1–MEG3 Locus.
Outline how the methylation states of imprinted genes change from sperm to adult?
Cheeky Huntely…
What we need to know about imprinting?
- What it is?
- How it is maintained - passed down
- Some examples of Genes that are imprinted
Why does imprinting even occur in the first place?
Is it possible that some epigenetics is transferred via RNA molecules?
Is epigenetics also found in plants?
Epigenetics in Plants
- Epigenetics is not just found in mammals, it is also common in flowering plants
- Plants depend on epigenetic processes for proper function
- The epigenetic modifications include DNA methylation, histone modifications, and the production of micro RNAs (miRNAs) that mediate epigenetic modifications
Difference between imprinting and inherited methylation?
Lecture 5 conclusions?
