Gene expression analysis Flashcards

1
Q

Define gene expression

A

The process by which the information encoded in a gene is turned into a function. This mostly occurs via the transcription of RNA molecules that code for proteins or non-coding RNA molecules that serve other functions

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2
Q

What is the use of mRNA expression measurements

A
  • Estimate environmental or genetic effects of phenotypes
  • Find differences in gene expression that could explain differences in phenotypes
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3
Q

What do you need to complete a PCR?

A
  • Template DNA
  • Heat stable polymerase
  • Water
  • Oligonucleotides (primer)
  • Precise thermocycling
  • Nucleotides
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4
Q

What is step 1 of PCR

A

Denaturation
- water with DNA in there
- 90 degrees
- 1 min
- DNA splits into single strands

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5
Q

What is step 2 of PCR

A

Annealing
- Cool whole thing down
- Add primers that bind to complementary DNA
- 54 degrees
- 45 seconds

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6
Q

What is step 3 of PCR

A

Extension
- Heat up again
- Add nucleotides
- 72 degrees
- 2 mins

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7
Q

At what cycle of PCR do you get target DNA

A

not until the 3rd cycle.
This is because you dont want any DNA with longer tails than the initial DNA

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8
Q

What makes a good primer?

A
  • Unique in the genome
  • lack of secondary binding sites
  • Tm = 52-65 degrees
  • Absence of dimerization capability
  • Low specific binding at the 3’ end
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9
Q

Describe uniqueness in terms of PCR primers

A
  • There shall be only one target site in the template DNA where the primer binds
  • There shall be no annealing site in possible contaminant sources
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10
Q

Describe why the length of the primer is important

A
  • effects the uniqueness and the Tm of the primer
  • Longer the primer the more chance it is unique
  • HOWEVER - the longer the primer the higher the annealing temperature - bad
  • Typically 17-28 bases long
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11
Q

Why is base composition important in a primer

A
  • High GC content means binds strong, this is because GC=3H bonds and AT=2H bonds
  • It effects the hybridisation specificity and annealing temp
  • There should be no long AT/GC regions
  • Usually GC content = 50-60%
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12
Q

Why is melting temperature important in primers

A
  • Tm is the temp at which half the DNA strands are single stranded and half are double stranded
  • High GC=High Tm because of increased H bonds
  • Shorter than 13 bp = Tm = (wA x xT)x2 + (yG + zC)x4
  • Longer than 13 bp = Tm = 64.9 + 41 (yG + zC - 16.4)/(wA+xT+yG+zC)
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13
Q

Why is annealing temperature important in primers

A
  • The temperature at which primers anneal to the template DNA
  • Tanneal = Tm-4
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14
Q

Why are primer secondary structures bad

A
  • If primers anneal to themselves or anneal to each other rather than the template, the PCR efficiency will decrease
  • Hairpin, Delf-dimer and dimer
  • Can be harmless if annealing temp doesn’t allow them to form
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15
Q

What is the max difference in annealing temp between the forward and reverse primer?

A

3 degrees

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16
Q

What are the main points to remember in primer design

A
  1. Unique
  2. Length - 17-28 bases
  3. Base composition - GC- 50-60%
  4. High stability 5’ end, low stability 3’ end
  5. Tm = 55-80 degrease
  6. Primer sets to anneal 2-3 degrees of each other
  7. Minimize secondary structure
17
Q

What are the primer design softwares?

A
  • Ensembl - sequence data
  • Primer3 - Primer design
  • Repeat masker - Mask repeat sequences
  • PrimerBLAST - check for off target effects
18
Q

Briefly describe rt-qPCR

A
  • Design 2 primers for gene of interest
  • Design 2 primers for the housekeeping gene
  • Relative expression of gene x vs hk gene
  • small no of genes
  • large no of samples
  • very cheap
  • all values are relative
19
Q

What is the housekeeper gene?

A
  • Genes that encode for proteins that are essential for cellular function
  • Often remains constant under most experimental conditions
  • Common housekeepers:
  • ACTB
  • GAPDH
    -rRNA
  • UBC
20
Q

What are the steps for the analysis of qPCR

A
  • Compute the mean of the technical replicates
  • Compute delta CT
  • Compute mean delta CT for the control
  • Compute delta delta CT
  • Compute 2-delta delta CT
21
Q

How do you know that output of qPCR is reliable?

A
  • delta CT values reliable when
    <0.3 away from the median of the group
22
Q

What do you do if the control is not reliable

A

Use the geometric mean
If there is a single outlier remove the offending replicate
Otherwise redo the experiment

23
Q

What are micro-arrays?

A
  • collection of microscopic DNA spots of a slide
  • Each spot have DNA known as probes or reporters
  • Target fluorophore - labelled target cDNA is hybridised in the arrays
  • Hybridisation is detected by intensity, giving a relative abundance of target sequence
24
Q

Describe the micro-array workflow

A

Sample
Purification
RT - reverse transcriptase to get only DNA not RNA
Coupling
Hybridization and washes
scanning
normalization
analysis

25
What is hybridization
Complementary nucleic acid sequences pair with each other by forming H bonds between complementary base pairs More complementary base pairs in a nucleotide sequence means tighter non-covalent bonds between the strands
26
What are the types of microarrays?
1-channel - provides intensity data for each probe or probe set indicating a relative level of hybridization with the labelled target 2-channel - typically hybridized with cDNA prepared from two samples to be compared. labelled with Cy3 (green and Cy5 (red). Yellow if there is no difference. Normal cells expressing higher = green. Cancer cells expressing higher = red
27
What are some applications of micro-arrays
- Comparative hybridization - DNA comparison - Expression profiling - SNP genotyping - ChIP on chip
28
What is the microarray workflow
create oligo arrays Extract RNA RNa to DNA Cy3 and 5 labelling hybridisation scanning data storage expression levels normalisation expression clustring interpretation
29
Normalisation is used to compensate for
varying behavior of dyes variations during the hybridisation variation in the manufacturing of the microarrays
30
What can go wrong with micro arrays
fibres scratches air bubbles spatial bias background haze
31
Describe background correction
adjust for non-specific hybridisation Using exogenous negative control spots
32
Describe gene-expression profiling
- Goal - to identify the genes that are differently expressed between groups - T-test, Anova, RankProducts
33
Describe type 1 and type 2 error
1 - calling a gene significantly changed even if its just by chance - avoided by Bonferroni correction 2 - missing a significantly changed gene - avoid by Benjamin-Hochberg false discovery procedure