Control and Measure of Gene Expression Flashcards
1
Q
Northern Blot
A
- RNA extraction
- electrophoresis
- northern blotting (RNA transfer to membrane)
- membrane hybridized with labelled probes and visualised
- more RNA = more probes = darker bands
- use both target gene and internal control gene to normalise RNA levels
- poor accuracy levels (visualises relative change)
2
Q
PCR
A
- using probes specific for the genes being measured the number of copies are directly proportional to the initial mRNA levels
3
Q
qPCR
A
- use of a reporter probe binding to target DNA if present
- as taq polymerase replicates the DNA the probe is destroyed releasing the fluorophore
- level is quantified
- threshold cycle shows the number of cycles taken to detect a real signal
4
Q
qPCR normalisation
A
- compensate for initial variations in mRNA and technical sequencing differenecs
- measure control and normalise against experimental
- Ct values used to normalise
5
Q
Limitations of Northern Blot and qPCR
A
- limited in number of genes tested at a time
- limited info (only tell you a gene is expressed)
- blot not quantitatively accurate
6
Q
Microarrays
A
- high throughput
- detects thousands of genes simultaneously
- relies on base pairing hybridisation with probes for each gene to be measured
- more hybridization gives higher fluoresence
- can measure differing expression over time, between tissues, co-expression, etc
7
Q
Affy GeneChip
A
- each genes has 16-20 pairs of probes synthesized on the chip
- individual probe for each gene
- control and experimental samples hybridized to separate slides and compared
- expression fold change calculated by comparasion
8
Q
Limitations of Microarrays
A
- image distortions or light merging
- may need statistical manipulation
- if you have multiple probes for the same gene giving different readouts = problem
- probes unavailable for all genes
- noisy data
- low expression genes not detected
- no information about which gene transcript is expressed
9
Q
RNA sequencing
A
- uses next gen sequencing technology to measure expression
- assumes every mRNA is sequenced the same number of times (if experiment show 2x as much mRNA vs control the expression is 2x more)
10
Q
Benefits of RNA seq
A
- accurate measure of expression, even at low levels
- can identify transcript
- identify novel transcripts with novel splice sites
11
Q
RNA seq Method
A
- mRNA isolation and conversion to cDNA
- sequencing adaptors added
- illumina sequencing
- alignment against genome
- generate sequence counts for all genes in genome
- read counts proportional to gene expression level
12
Q
RNA seq normalisation
A
- compensate for initial variations in mRNA and technical differences with sequencing
- scales read counts so they can be compared
1. raw read count normalisatoin
2. reads/fragments per kilobase per million reads
13
Q
Raw Read Count Normalisation
A
- aim to make normalised count for non differentially expressed genes similar between samples
- doesn’t adjust count distributions between samples
- assumes most genes not differentially expressed adn differentially expressed genes divided equally between up and down
- divide gene count by geometric mean and take median
- apply this normalisation factor
14
Q
RPKM/FPKM
A
- normalises for gene length and library size
15
Q
Transcriptome Profiling
A
- identify variable transcripts where reads cross exon boundaries
- microarrays can’t do this
- reads map to previously annotated sites and novel expressed sequences
16
Q
Single Cell RNA seq
A
- normal RNA seq confounded by heterogeneity of sample
- different cell types, mutations, cell cycle stages, etc
- can analyse single cells for improved resolution and identification of heterogenity in populations
- however: gives list of likely/probable expression only
17
Q
DNA methylation
A
- reversbile methylation of cytosine by methyltransferase
- occurs at CpG sites
- silences gene expression: prevents TF binding, modifies chromatin structure
- Cpg islands can identify promoter regions as methylation increases risk of mutation to T (promoter regions under stringent control so less likely to mutate)
18
Q
Epigenetics
A
heritable changes in gene expression or phenotype not caused by nucleotide changes
19
Q
Methylation Maintenance
A
- hemi methylated DNA recognise by DNMT1 methylating the new strand to maintain the methylation state
20
Q
Methylation and Disease
A
- methylation patterns differ in many diseases
- implicated in cancer
- hypomethylation at intergenic repeats and repeats gives genomic instability
- genome wide in every cancer
21
Q
Locations of DNA methylation
A
- intergenic regions
- repetitive region
- gene upstream regions (non methylated)
- promoter regions usually unmethylated and create CpG islands at higher density due to selective pressure
22
Q
Intergenic Regions
A
- methylation maintains genomic integrity by forming compacted chromatin/less accessible
- silences abberrant promoters or splice sites
23
Q
Repetitive Elements
A
- transposable elements are highly mutagenic
- methylation stops this
- methylation of promoters silences repeat and mutates into T so prevents transcription
- prevents recombination
24
Q
MeDIP-Seq
A
- methylated DNA immunoprecipitation
- DNA denatured and fragmented
- antibody used to separate methylated
- immunoprecipitation separates
- isolated methylated regions sequenced via next gen sequencing
- sequences mapped onto reference genome
25
Q
Bisulfite Sequencing
A
- samples treated with bisulfite converting cytosine to uracil
- doesn’t happen in methylated cytosine
- treated samples sequenced and compared to determine methylation
- great resolution
26
Q
X Inactivation
A
- silencing of one of the X chromosomes in female mammels
- dosage compensation avoiding gene over expression
- inactivated chromosome compacted by histone methylation
- Xist gene triggers this (long non-coding RNA)
27
Q
Long Noncoding RNA
A
- ncRNA over 200bp
- function largely unknown
- target gene transcription and provide a system of complex control of expression in eukaryotes
28
Q
Xist
A
- lncRNA
- expressed from only one of the two X chromosomes
- first detectable event in X inactivation
- expression determines inactivated chromosome
- Xist RNA coats inactive X (cis)
- contains many repeats within transcript
- Repeat A has silencing function binding histone methyltransferase complex laying down methylation
- Xist has sequence specificity for X guiding the complex along chromosome
29
Q
HOTAIR
A
- acts as a guide and also a scaffold
- expressed from HOXC locus on chromosome 12 and represses the HOXD locus on chromosome 2
- acts in trans
- binds to PRC2 (adding repressive histone methylation) and LSD1 (removing active histone methylation) to produce repressive chromatin structure
30
Q
Identifying Histone Modification
A
- similar to MeDIP-Seq
- fractionate DNA
- use antibody binding only to modified histone
- immunoprecipitation for separation
- sequence isolated fraction
- map onto genome to identify regions with modified histones and therefore genes under regulation
31
Q
Transcription Factors
A
- stabilise or block RNAP binding
- recruit coactivator and corepressor proteins to complex
- catalyse acetylation/deacetylation of histones
- acetylation weakens association with DNA increasing accessibility (and vice versa)
32
Q
ZNF143-Notch
A
- when ZNF143 is bound RBPJ can’t bind : ZNF controls accessibility of RBP sites to the Notch/RBP complex
- dissociation allows sites to be occupied by RBP complexes (repression)
- notch presence allows transcriptional activation
33
Q
ChIP-Seq
A
- uses known binding protein to identify binding regions
- immunoprecipitation enriches complexes containing target protein
- directly sequences the bound DNA and maps back onto genome for localisation
- most frequently sequences fragments form coverage peaks at specific locations
