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Genomics > Microarrays > Flashcards

Microarrays Flashcards

1
Q

What is a microarray?

A

An ordered assembly of nucleic acids immobilised on a solid support

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2
Q

Why is DNA used in microarrays rather than RNA?

A

Typically short DNA nucleic acid sequences are immobilised as its much more robust than RNA
Structurally it’s much easier to predict - easier to make

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3
Q

Outline the process of microarrays

A
  1. ssDNA or RNA sequences
  2. Label sequences with fluorescent tags
  3. Hybridise the strands
  4. Detect which strands are brighter / darker
    => gives idea of gene expression
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4
Q

How does microarraying work?

A

Lots of copies of the same probe in a spot
Each spot gives the relative expression for one transcript
Detects all known transcripts in one sample

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5
Q

What does each spot represent?

A

Image contains red, green and yellow spots.

Each spot represents one SNP

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6
Q

What are the pros of carrying out microarrays?

A

Because we have lots of spots we can analyse lots of spots simultaneously
And microarrays therefore allow us to analyse genetic markers across the genome

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7
Q

Outline the expression profiling workflow

A

Gene activity 🡪 transcript level 🡪 signal

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8
Q

How is gene expression profiling carried out before analysis can occur?

A
  1. Extract RNA from sample
  2. Purify sample by removing ribosomal RNA/DNA
  3. Label sample with fluorescent tags
  4. Hybridise samples
  5. Detect signal and analyse
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9
Q

Outline the steps of microarray data analysis

A 
Normalisation 
Heirarchical Clustering
Gene Filtering 
Statistical Tests 
Generate Gene List 
Biological Interpretation
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10
Q

What is normalisation?

A

Normalisation: making sure there aren’t any probes binding preferentially other than the fact they’re expressed
E.g. non-specific binding

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11
Q

What is the most common Microarray data analysis technique?

A

Hierarchical clustering is the core analysis technique here

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12
Q

What is shown by pathway analysis?

A

Pathway analysis shows whether genes are up/down regulated

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13
Q

What is clustering?

A

Organises data with similar patterns into classes

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14
Q

What do the classes in clustering show?

A

Objects within a class are more similar to each other than to objects outside the class

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15
Q

What are dendograms?

A

Dendrograms - ‘trees’
Alternative way of displaying similarity between samples

Distant samples are less similar

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16
Q

What is the significance of data repositories?

A

Microarray experiments aren’t cheap, so maximise utility

  • share data
  • use other people’s data
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17
Q

What is the MIAME?

A

Minimum Information About a Microarray Experiment (MIAME) - makes it easier to compare results

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18
Q

What is NICE?

A

National Institute for Health and Care Excellence

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19
Q

What tests are used to predict Cancer recurrence?

A

Mammaprint, Oncotype DX, IHC4 and Mammostrat

20
Q

How does mammaprint work?

A

Mammaprint uses microarrays to study 70 genes (standard) thought to be associated with the cancer

  • Fast
  • Know what looking for
21
Q

What is the benefit of mammaprint microarray?

A

20% more low risk breast cancer patients have been identified using this technique - vast majority no longer receive adjuvant chemotherapy which is beneficial to patients and is more cost effective for nhs

22
Q

Outline the central dogma of biology

A

DNA –>RNA –> Protein

23
Q

What is reverse transcriptase?

A

Enzyme used by retroviruses to convert their RNA genomes into DNA

24
Q

What is reverse transcriptase used for in the lab?

A

convert RNA into cDNA – complementary DNA

We can perform PCR on cDNA and run the products on a gel

25
Q

What is a housekeeping gene?

A

gene expressed in all tissues at a similar level (e.g. GAPDH, beta-actin)

26
Q

How is RT-PCR made quantitative?

A

We can make RT-PCR quantitative by counting the number of copies of amplified DNA present.

27
Q

What does the Ct value tell us?

A

Fluorescence above background at 225 copies = Ct

28
Q

What does a low Ct value show?

A

The higher the amount of starting RNA (cDNA),

the lower the Ct value

29
Q

What are the 2 fluorescence ways to count the copies produced in PCR

A
  1. Fluorescent dye

2. Label probes

30
Q

Explain how the fluorescent dye works

A

Include a dye in the PCR reaction mix that fluoresces when it binds double-stranded DNA, e.g. an intercalating dye such as SYBR Green

31
Q

Describe how labelling probes with fluorescent tags allows them to be detected

A

Label a probe in the PCR that only fluoresces when it is incorporated in the PCR product, e.g. TaqMan

32
Q

What are intercalating dyes?

A

Intercalating dyes are so-called because they bind between the stacked DNA base pairs

33
Q

What is qPCR used for?

A

qPCR is used to independently confirm differences in RNA levels between samples

34
Q

Why is qPCR a better method to use than probe binding?

A

Probe binding is noisy and differences can be detected that are not real, especially where differences are small (<2-fold)

35
Q

What are the pros and cons of using qPCR?

A

RNA-Seq is a more accurate measure of RNA transcript abundance, it is more reproducible and works over a wider range of concentrations…..but it is more expensive

36
Q

What enables us to carry out GWAS?

A

Genome-wide Association Studies are only possible because we can genotype large numbers of SNPs in large numbers of subjects

37
Q

How are large numbers of SNPs genotyped?

A

This is possible by using microarrays that hybridise with genomic DNA adjacent to SNPs (rather than RNA transcripts)

38
Q

How are the SNPs detected in SNP microarrays?

A

The SNP is then extended by one base that is fluorescently labelled and detected using a high definition scanner

39
Q

What does a spot contain?

A

In a spot there are lots of copies of the same single-stranded oligonucleotide – a “probe”

40
Q

What is a probe used for?

A

Each probe is for genotyping one SNP

41
Q

What is an oligonucleotide?

A

a single stranded piece of DNA approximately 20-30 nucleotides long

42
Q

How are SNP Genotypes translated by software?

A

Software translates the three different colour signals for each probe into genotypes (homo- and heterozygous)
A few SNPs are reviewed by hand (<50) but most are not

43
Q

What are copy number variants?

A

Typically defined as sequences greater than 1kb that have different copy numbers in different people

44
Q

How often do copy number variants occur in the genome?

A

> 2000 identified
10kb-5000kb
repeated/absent
~12% genome = CNV

45
Q

How many copy number variants copies are present in most individuals?

A

Most people have 2 copies of these CNVs (one from each parent), some have 1 or none or several <5 or 10 copies.
More common than SNPs

46
Q

How often do structural variants occur?

A

Lots of versions of structural variants which can also be copy number variants

47
Q

How does array comparative genomic hybridisation allow us to see visualise the results?

A

The output of the scanning process is the log of the ratio of the fluorescence intensities for each spot – usually this is 0 as there are equal amounts of red and green signal.

Genomics (20 decks)

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