Gene Expression Analysis Flashcards
Complete deck for lecture 3 (106 cards)
1
Q
At which two main stages can gene expression be controlled?
A
Transcriptional (is gene on or off?)
Translational (is mRNA being turned to protein?)
2
Q
What are the three parameters of gene expression?
A
Level of regulation
Time / circumstances when expressed
Place / location / cells / tissues / organs expressed in
3
Q
What can we analyse to investigate transcriptional control?
A
mRNA accumulation for gene
4
Q
What can we analyse to investigate translational control?
A
Protein accumulation for gene
5
Q
What might be useful about knowing the location in the body a gene is expressed more in?
A
Could be useful for determining function
6
Q
What techniques are used for measuring mRNA levels?
A
Northern blotting
RT-PCR
Microarray (GeneChip) techniques (genomic level)
7
Q
What techniques are used for measuring protein levels?
A
Western blotting
8
Q
When is a label incorporated into a probe?
A
When the probe is synthesised
9
Q
List the three main types of probe
A
Radioactive
Chemical
Fluorescent
10
Q
How are radioactive probes detected?
A
Autoradiography
11
Q
Compare a 32P probe with 35S, 14C and 3H probes
A
32P has high energy, easy detection and is sensitive
35S, 14C and 3H have lower energies, are less sensitive, but have higher resolution
12
Q
List some different radioactive probes
A
32P
35S
14C
3H
13
Q
How do chemical probes work?
A
Incorporate an antigen (AKA recognition signal for an enzyme linked to an antibody) into DNA
Makes a coloured or chemiluminescent compound
14
Q
How do fluorescent probes work?
A
There are many different fluorescent compounds available to be incorporated into nucleotides
Detected with specialised equipment
15
Q
What is a disadvantage of using fluorescent probes?
A
You need specialised equipment to detect the signal
16
Q
How and where are labelled nucleotides incorporated into DNA?
A
Via DNA polymerase, and in vitro
17
Q
What is a probe?
A
A small piece of single-stranded, synthetic, labelled DNA, complementary to the sequence of interest so it can base pair with it and make it detectable
18
Q
Why must 32P probes be handled with care?
A
The high energy could damage human cells
19
Q
Fill in the gaps:
A probe is a small piece of __________, _______, _______ DNA, complementary to ____________________ so it can base pair with it and make it _________.
A
A probe is a small piece of single-stranded, synthetic, labelled DNA, complementary to the sequence of interest so it can base pair with it and make it detectable
20
Q
Give two differences between the procedures for Northern blotting and Southern blotting
A
In Northern blotting:
mRNA is in our membrane instead of DNA
DNA probe forms a DNA RNA double strand which is stable for detection once in the membrane
21
Q
True or False:
Northern blotting is more common than reverse transcriptase PCR (RT-PCR)
A
False
Reverse transcriptase PCR (RT-PCR) is more common than Northern blotting
22
Q
True or False:
Reverse transcriptase PCR (RT-PCR) is more common than Northern blotting
A
True
23
Q
Why did RT-PCR replace Northern blotting for routine analysis?
A
It is simpler, quick and cheap
24
Q
What do we use RT-PCR and Northern blot to measure?
A
The mRNA levels, which indicate how much a gene is expressed
25
What does mRNA accumulation for gene indicate?
Transcriptional control, how much a gene is expressed
26
What does protein accumulation for gene indicate?
Translational control, how much the mRNA is being converted into protein
27
What does RT-PCR stand for?
Reverse transcriptase - polymerase chain reaction
Or sometimes, Real Time PCR (which is different?)
28
Fill in the gaps: cDNA formation 1/2
The _____ tail in _____ end of mRNA in eukaryotes is used as an ______ for _______ ________ DNA polymerase.
The provided _____ primer base pairs with the complementary _____ tail, forming a double strand.
The double strand is an _____ point for the _____ DNA polymerase to generate a double strand molecule of DNA, forming an _______ ____ ____ _______.
The poly-A tail in 3’ end of mRNA in eukaryotes is used as an anchor for reverse transcriptase DNA polymerase.
The provided poly-T primer base pairs with the complementary poly-A tail, forming a double strand.
The double strand is an anchor point for the 5’ - 3’ DNA polymerase to generate a double strand molecule of DNA, forming an unstable DNA RNA hybrid.
29
Fill in the gaps: cDNA formation 2/2
The provided ______ degrades the RNA.
The provided ______ _________ builds a new anchor for the ____ __________ to extend, using the single DNA strand as a template and generating _______ _________ DNA.
The new DNA molecule is stable and corresponds to the original _____, the cDNA is complete.
The provided RNAse degrades the RNA.
The provided terminal transferase builds a new anchor for the DNA polymerase to extend, using the single DNA strand as a template and generating double stranded DNA.
The new DNA molecule is stable and corresponds to the original mRNA, the cDNA is complete.
30
Fill in the gaps: RT-PCR amplification
The _____ is _________ to get single strands of the template. The provided gene-specific ______ base pair with the _____ __ _______ within the total mix of the DNA. Using PCR the template DNA for the _______ ____ is amplified to give many more copies of itself.
The amount of copies of cDNA for the gene of interest at the end depends on _______________________. This depends on how much ______ was present for that gene when making the _____, which depends _________________________.
The cDNA is denatured to get single strands of the template. The provided gene-specific primers base pair with the gene of interest within the total mix of the DNA. Using PCR the template DNA for the specific gene is amplified to give many more copies of itself.
The amount of copies of cDNA for the gene of interest at the end depends on how much cDNA for that gene was present initially. This depends on how much mRNA was present for that gene when making the cDNA, which depends on how much the gene was being expressed.
31
True or False:
| A low number of copies of cDNA for the gene of interest after PCR indicates that the gene is highly expressed.
False
| A high number of copies of cDNA for the gene of interest after PCR indicates that the gene is highly expressed.
32
True or False:
| A high number of copies of cDNA for the gene of interest after PCR indicates that the gene is highly expressed.
True
33
How are the products of RT-PCR amplification detected?
Via nucleic acid gel electrophoresis.
Intensity of the band indicates how much a gene is expressed (No band means gene is not expressed at all, faint band means lowly expressed gene, strong band means highly expressed gene)
34
After RT-PCR amplification, what does no band on the nucleic acid gel electrophoresis mean?
No band means no mRNA was present at the beginning so the gene is not expressed at all.
35
After RT-PCR amplification, what does a faint band on the nucleic acid gel electrophoresis mean?
A faint band means there was very little mRNA present at the beginning so the gene is lowly expressed.
36
After RT-PCR amplification, what does a strong band on the nucleic acid gel electrophoresis mean?
A strong band means there was a lot of mRNA present at the beginning so the gene is highly expressed.
37
What are the stages of RT-PCR? (Briefly)
1. Isolate total mRNA from cells/tissue of interest
2. Generate cDNA (see previous slide)
3. Use PCR to amplify cDNA of target gene
4. Gel Electrophoresis for results
38
What is different about the process of quantitative real time RT-PCR in comparison to normal RT-PCR?
Mostly the same as RT-PCR with extra steps:
At PCR stage, incorporate fluorescent label (most common is Cybergreen)
At the end, can quantify fluorescent signals and correlate to original mRNA levels/gene expression level
39
What are the disadvantages of quantitative real time RT-PCR (qPCR)
Requires specialised equipment
| Expensive
40
What are the advantages of quantitative real time RT-PCR (qPCR)
More accurate results (technique for measuring exact quantification of gene expression)
Efficient technique
Can be used after each PCR cycle
41
What is the most common fluorescent label to incorporate into the cDNA in quantitative real time RT-PCR?
Cybergreen
42
What do microarray assays and gene chips allow us to do?
Measure expression of thousands of genes at a time
Compare gene expression patterns for thousands of genes at different times, in different tissues, or under different conditions
Simultaneous analysis of thousands of different mRNAs by hybridisation with labelled complementary cDNA
43
What is a Microarray? (With specific examples)
A set of DNA fragments attached to a glass microscope slide
DNA fragments could be cDNA, gene fragments or oligonucleotides
44
What is a Gene Chip?
A collection of DNA oligonucleotides on a stamp-sized chip, manufactured using photolithography
45
What is an advantage of earlier developed tools such as Microarrays or Gene Chips, in comparison to Genomics or sequencing total RNA / RNAseq?
They are cheaper and can still analyse global gene expression
46
Fill in gaps: hybridisation microarrays / gene chips 1/2
The same principle for analysing a single gene with fluorescent probes.
1. Isolate _____ _____ of tissue / cells we are interested in analysing
2. Make _____ from total _____. During _______ ____________ label cDNA with __________ nucleotide so that single stranded _________ template cDNAs are generated.
Alternatively, generate multiple fluorescently labelled ______ __________, covering the whole genome.
Make sure label is ________ for the samples of _______ sources.
3. Labelled cDNA or oligo nucleotides applied to slide or chip where multiple copies of ______ _________ ____ __________ from the organisms genes are fixed.
The same principle for analysing a single gene with fluorescent probes:
1. Isolate total mRNA of tissue / cells we are interested in analysing
2. Make cDNA from total mRNA. During reverse transcription label cDNA with fluorescent nucleotide so that single stranded fluorescent template cDNAs are generated.
Alternatively, generate multiple fluorescently labelled oligo nucleotides, covering the whole genome.
Make sure label is different for the samples of different sources.
3. Labelled cDNA or oligo nucleotides applied to slide or chip where multiple copies of single stranded DNA fragments from the organisms genes are fixed.
47
# Fill in gaps: hybridisation microarrays / gene chips 2/2
4. In the slide, each spot has ________ _____ of a unique DNA fragment corresponding to a _______ _____. Each spot has a _________ fragment so tests for a _________ _____.
5. The fluorescently labelled ______ from the samples are ______ _________, then put in each well. They will hybridize with any cDNA / oligo nucleotides in the microarray / gene chip they are ____________ to.
6. Excess labelled cDNA is then ___________.
7. Microarray scanned for __________. Each ___________ spot represents _ _____ __________ in ____________ of the samples.
4. In the slide, each spot has multiple copies of a unique DNA fragment corresponding to a specific gene. Each spot has a different fragment so tests for a different gene.
5. The fluorescently labelled cDNAs from the samples are mixed together, then put in each well. They will hybridize with any cDNA / oligo nucleotides in the microarray / gene chip they are complementary to.
6. Excess labelled cDNA is then washed off.
7. Microarray scanned for fluorescence. Each fluorescent spot represents a gene expressed in one or more of the samples.
48
What is the process of hybridisation for microarrays / gene chips? (Whole thing)
The same principle for analysing a single gene with fluorescent probes:
1. Isolate total mRNA of tissue / cells we are interested in analyzing (e.g. tumour and normal cells)
2. Make cDNA from total mRNA. During reverse transcription label cDNA with fluorescent nucleotide so single stranded fluorescent template cDNAs are generated.
Alternatively, generate multiple fluorescently labelled oligo nucleotides, covering the whole genome. Make sure label is different for the samples of different sources.
3. Labelled cDNA or oligo nucleotides applied to slide or chip where multiple copies of single stranded DNA fragments from the organisms genes are fixed.
4. In the slide, each spot has multiple copies of a unique DNA fragment corresponding to a specific gene. Each spot has a different fragment so tests for a different gene.
5. The fluorescently labelled cDNAs from the samples are mixed together, then put in each well. They will hybridize with any cDNA / oligo nucleotides in the microarray / gene chip they are complementary to.
6. Excess labelled cDNA is then washed off.
7. Microarray scanned for fluorescence. Each fluorescent spot represents a gene expressed in one or more of the samples.
49
Fill in the gap:
| Most microarray experiments will compare __ sets of mRNA samples.
Most microarray experiments will compare 2 sets of mRNA samples
50
In a microarray experiment comparing only two sets of mRNA samples, what are the two samples?
How are they labelled?
Controlled, untreated sample
Experimental sample
They are each labelled with a different colour fluorescence
51
How are the results of a microarray interpreted?
The intensity of the fluorescence at each spot correlates with the expression of that particular gene.
The colour of the spot is linked to relative expression levels of that particular gene in the two samples.
52
In a microarray what does the intensity of fluorescence at each spot indicate?
The expression of that particular gene
53
In a microarray what does the colour of each spot indicate?
Give examples of what you would see in different scenarios.
The relative expression levels of that particular gene between the two samples
(Eg; if it's completely the colour associated with the control, expression in control only. If it's completely the colour associated with the experimental sample, expression in experimental sample only. If it's a hybrid, expression in both. Black indicates no expression in either.)
54
What does a black spot indicate in a microarray?
No expression in control sample or experimental sample(s)
55
What does a hybrid coloured spot indicate in a microarray?
Expression in multiple samples
56
What does a single coloured spot indicate in a microarray?
Expression in only one sample
57
Why is analysing expression of many genes evaluated at the same time as each other, under a specific condition, useful?
Allows us to group genes based on their patterns of expression
58
What are 'clustered' genes?
A group of genes expressed in a coordinated manner working in pathways active at the same time/under the same condition, which are therefore likely to be related in function
59
True or False:
Each dot on a microarray is a well containing identical copies of DNA fragments that carry a specific gene, and each well contains different DNA fragments to the others.
True
60
Why is transcriptome cluster analysis useful?
Analysing a full set of genes together, over time, can help identify genes with potential co-regulation when multiple genes behave the same way.
Helps understand networks of genes possibly working together, OR processes occurring in the same temporal bases.
61
Define transcriptome
The sum total of all the messenger RNA molecules expressed from the genes of an organism
62
In a transcriptome cluster analysis, what does red mean?
Gene has been downregulated
63
In a transcriptome cluster analysis, what does green mean?
Gene has been upregulated
64
True or False:
| All of the dots on a microarray are wells which contain identical copies of DNA fragments to each other.
False
Each dot on a microarray is a well containing identical copies of DNA fragments that carry a specific gene, and each well contains different DNA fragments to the others.
65
What does ISH stand for?
In situ hybridisation
66
What does in situ hybridisation allow us to study?
The precise time and location of mRNA in a cell or tissue or whole organism (unicellular organism or multicellular embryos) in a histological section.
67
What form of probe is usually used for in situ hybridisation (ISH)?
Fluorescent
| Can be called Fluorescent in situ hybridisation (FISH)
68
True or False:
| It is not possible to use a radioactive probe for in situ hybridisation.
False
| It is possible to use a radioactive probe for in situ hybridisation.
69
In in situ hybridisation, what does the probe couple to and why?
A reporter molecule, to localise the RNA or DNA in the histological section
70
True or False:
In in situ hybridisation, the probe couples to a repeater molecule to release the RNA or DNA from the histological section
False
In in situ hybridisation, the probe couples to a reporter molecule to localise the RNA or DNA in the histological section
71
True or False:
| It is possible to use a radioactive probe for in situ hybridisation.
True
72
What can in situ hybridisation be used for?
Analysing changes in abundance and patterns of distribution of mRNA helps indicate their function in organism.
Used in developmental biology, studying change in patterns of expression over different stages of development.
Used in karyotyping chromosomes.
73
What does epigenetic modification refer to?
What can it lead to?
Heritable changes in gene activity that are not the result of changes in DNA sequence.
Can lead to changes in gene expression and cellular phenotype.
74
What does the word 'epigenetic' mean, literally?
In addition to the genetic sequence
75
List 5 epigenetic processes
```
DNA methylation
Acetylation
Phosphorylation
Ubiquitylation
SUMOylation
```
76
Define epigenome
The complete description of chemical changes to DNA and histones and mapping the genome in a given cell type
77
What are the two most common epigenetic processes?
DNA methylation
| Acetylation
78
Why is more generally known about DNA methylation than the other epigenetic processes?
Methylation is the easiest to study with existing technology
79
What is DNA methylation?
Addition or removal of a methyl group, predominantly on cytosine bases
80
What is chromatin?
The complex of histones and DNA tightly bound together to fit inside the nucleus
81
What is heterochromatin?
Tightly folded chromatin which makes the genes harder to transcribe, decreasing expression
82
What is euchromatin?
More loosely folded chromatin which makes the genes easier to access and therefore transcribe, increasing gene expression
83
What is the name given to tightly folded chromatin?
Euchromatin
84
What is the name given to loosely folded chromatin?
Heterochromatin
85
How is chromatin modified?
Acetyl groups are added or removed (acetylation) and affect the chromatin structure
86
What is imprinting?
When one allele is silenced by an epigenetic process
87
When may imprinting cause problems?
If the expressed allele is damaged, or carries a variant which increases vulnerability, for example to microbes, toxic agents, or harmful substances.
88
True or False:
| Imprinting was first described in 1991 in corn
False
| Imprinting was first described in 1910 in corn
89
True or False:
| Imprinting in mammals was confirmed in 1910
False
| Imprinting in mammals was confirmed in 1991
90
True or False:
| So far ~80 genes in humans and ~600 in mice identified as can be imprinted
True
91
True or False:
| So far ~60 genes in humans and ~800 in mice identified as can be imprinted
False
| So far ~80 genes in humans and ~600 in mice identified as can be imprinted
92
List 5 factors that affect epigenetic mechanisms
```
Development (in utero, childhood)
Environmental chemicals
Drugs/pharmaceuticals
Aging
Diet
```
93
True or False:
| Imprinting in mammals was confirmed in 1991
True
94
True or False:
| A methyl group can activate or repress genes
True
95
What are histones? What are they for?
Proteins that DNA can wind around for compaction and gene regulation
96
True or False:
| Imprinting was first described in 1910 in corn
True
97
How are histones modified?
How does this alter gene expression?
Epigenetic factors bind to the histone 'tails', which alters the extent that DNA is wrapped around the histones, and therefore how accessible the gene is for transcription
98
True or False:
| A methyl group always represses gene expression
False
| A methyl group can activate or repress genes
99
True or False:
In in situ hybridisation, the probe couples to a reporter molecule to localise the RNA or DNA in the histological section
True
100
List 4 potential health consequences of improper epigenetic processes
Cancer
Autoimmune diseases
Mental disorders
Diabetes
101
True or False:
| It is possible to determine if a change in gene expression is due to an epigenetic change by sequencing the DNA
False
Epigenetic processes do not affect the DNA sequence, so it is not possible to determine if a change in gene expression is due to an epigenetic change by sequencing the DNA
102
How does 3rd Generation Sequencing work?
Uses single molecule-real time technology
Activity of DNA polymerase monitored in real time as it uses provided fluorescently-labelled nucleotides to extend the DNA chain.
Changes in activity can therefore be observed when it encounters modified bases in the DNA template.
Allows analysis of the epigenome.
103
What has improved the precision for mapping chromatin and DNA methylation in 3rd Generation Sequencing techniques?
Ultra high throughput DNA sequencing
104
What does most research in epigenetics investigate?
Epigenetics related to cancer
105
Fill in the gaps: Epigenetics and E.coli
In ____, an E.coli outbreak in _______ affected thousands of people. ___ people died and nearly _____ people suffered ______ ______. Sequencing found that the __________ _______, but not the ____ __________, had changed. The __________ likely contributed to the _____________ of what was previously thought not to be a pathogenic strain.
In 2011, an E.coli outbreak in Germany affected thousands of people. 50 people died and nearly 1000 people suffered kidney failure. Sequencing found that the methylation pattern, but not the DNA sequence, had changed. The epigenetics likely contributed to the pathogenicity of what was previously thought not to be a pathogenic strain.
106
Fill in the gaps:
Genome-wide catalogues of epigenetic control elements are now available for cells in different ______, as well as for associated __________ and ____________ __________. Serves for future studies to understand the role of epigenetic mechanisms to regulate _______ __________, _______ and _____.
Genome-wide catalogues of epigenetic control elements are now available for cells in different states, as well as for associated phenotypes and environmental conditions. Serves for future studies to understand the role of epigenetic mechanisms to regulate normal physiology, disease and aging.
