Control and Measure of Gene Expression Flashcards
(33 cards)
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
Bisulfite Sequencing
- 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
X Inactivation
- 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
Long Noncoding RNA
- ncRNA over 200bp
- function largely unknown
- target gene transcription and provide a system of complex control of expression in eukaryotes
28
Xist
- 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
HOTAIR
- 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
Identifying Histone Modification
- 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
Transcription Factors
1. stabilise or block RNAP binding
2. recruit coactivator and corepressor proteins to complex
3. catalyse acetylation/deacetylation of histones
- acetylation weakens association with DNA increasing accessibility (and vice versa)
32
ZNF143-Notch
- 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
ChIP-Seq
- 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
