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Antibody And DNA Technology > Expression Profiling > Flashcards

Expression Profiling Flashcards

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Biology 101 flashcards
1
Q

Define transcriptiome.

A

The set of all mRNA molecules in one cell or a population of cells.

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2
Q

When observing the transcriptiome, how can the expression of a gene indicative of function be analysed?

A

By the site of expression and the size/content of the transcripts.

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3
Q

What are the three methods of single gene analysis?

A
  1. Northern blotting.
  2. RT-PCR.
  3. In situ hybridisation.
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4
Q

What are the three methods of wider analysis of the transcriptiome?

A
  1. Expressed sequence tags.
  2. Microarrays.
  3. Sequencing.
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5
Q

State the purpose of Northern blotting.

A

Studying gene expression by detection of RNA (or isolated mRNA) in a sample.

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6
Q

In Northern blotting, how are RNA samples denatured?

A

At 90°C and using formamide.

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7
Q

In Northern blotting, how are RNA samples separated?

A

By size electrophoresis in a denaturing agarose gel.

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8
Q

In Northern blotting, what type of denaturing agarose gel is used?

A

Formaldehyde.

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9
Q

In Norther blotting, what happens to the RNA after separation?

A

It is transferred to a nylon membrane, crosslinked and then hybridised with a labelled probe.

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10
Q

In Northern blotting, how is mature mRNA isolated?

A

Only those with a poly(A) tail are isolated.

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11
Q

In Northern blotting, what are the three types of hybridisation probes?

A
  1. Radiolabelled/non–radiolabelled labelled DNA.
  2. In vitro transcribed RNA.
  3. Oligo-nucleotides.
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12
Q

In Northern blotting, what is the alternative probe to hybridisation probes?

A

Sequences with partial homology.

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13
Q

What four types of information can Northern blotting deduce?

A
  1. Sites of expression.
  2. Level of expression.
  3. Size of transcript.
  4. Alternative spliceforms.
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14
Q

State the purpose of RT-PCR.

A

Amplify specific mRNAs by means of gene specific primers.

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15
Q

What three pieces of information does RT-PCR provide?

A
  1. Sites of expression.
  2. Level of expression (semi-quantitative).
  3. Alternative spliceforms.
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16
Q

RT-PCR qualitatively detects gene expression through creation of what?

A

Complementary DNA (cDNA) transcripts from RNA templates.

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17
Q

In PT-PCR, what are the three types of primers?

A
  1. Random.
  2. Oligo (dT).
  3. Sequence specific.
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18
Q

In PT-PCR, how is the locus amplified?

A

By means of specific primers.

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19
Q

In digital RT-PCR, why is the sample partitioned?

A

So individual RNA molecules within the sample are loacalised within various separate regions.

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20
Q

In digital RT-PCR, what type of distribution of molecules do you assume?

A

Poisson.

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21
Q

In digital RT-PCR, what does 0 represent?

A

A negative result.

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22
Q

In digital RT-PCR, what does 0 represent?

A

A positive result.

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23
Q

What is the last step of digital RT-PCR?

A

Absolute quantitation of number of copies of target.

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24
Q

How are tissues prepared for in Situ hybridisation?

A

They are cut by a microtome and then either frozen or embedded in wax.

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25
Q

During in Situ hybridisation, why is hybridisation of a suitable gene-specific probe to the tissue use?

A

To provide detailed expression images representative of the distribution of the RNA in the tissue.

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26
Q

What is the purpose of whole-mount in situ hybridisation?

A

To hybridise intact tissues or embryos.

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27
Q

In Whole-mount in situ hybridisation, what are the probes labelled with?

A

UTP-digoxigenin.

28
Q

In Whole-mount in situ hybridisation, what affects the permeability of the cells in order to open the cell membrane?

A

Proteinase K.

29
Q

In Whole-mount in situ hybridisation, the labelled probe binds to what substance?

A

mRNA.

30
Q

In Whole-mount in situ hybridisation, what happens to the cell after the probe has been bound?

A

It is washed.

31
Q

In Whole-mount in situ hybridisation, what type of antibody is used?

A

Alkaline phosphate-conjugated antibody.

32
Q

In Whole-mount in situ hybridisation, what does the antibody bind to?

A

Digoxigenin.

33
Q

In Whole-mount in situ hybridisation, how does the dye process work?

A

A colourless compound is added to the cell, which become purple when phosphate is removed.

34
Q

Define differential display.

A

Identifying changes in expression in mRNA transcripts between two cell samples.

35
Q

What two methods does differential display entail?

A

Partially degenerate PCR primers and modified RT-PCR for studying the expression of numerous genes simultaneously.

36
Q

State an advantage of using differential display.

A

There is no need for previous information about expressed genes.

37
Q

Define ESTs.

A

Small fragments of DNA sequence which is generated by sequencing either one or both ends of an expressed gene.

38
Q

How many nucleotides long are ESTs?

A

200 to 500.

39
Q

What are two advantages of ESTs?

A

Random and unbiased.

40
Q

ESTs can reconstruct complete sequences of what?

A

cDNA.

41
Q

What four pieces of information can be derived from ESTs?

A
  1. The cells where the gene is expressed.
  2. Level of transcripts (semi-quantitative).
  3. Isoforms (alternate promoter usage and alternative splicing).
  4. Homologs in other species.
42
Q

What does dbEST (Genbank) stand for?

A

Database of EST sequences.

43
Q

What is the purpose of Unigene within Genbank?

A

It automatically separates sequences into a non-redundant set of gene-oriented clusters.

44
Q

What are the two types of DNA chips?

A

cDNA (spotted by robot) and oligonucleotide (in situ synthesis by photolithography).

45
Q

State the four steps of microarrays.

A
  1. Isolate mRNA from tissue.
  2. Convert mRNA to first-strand cDNA.
  3. Degrade mRNA with NaOH and label cDNA.
  4. Hybridise fluorescent labelled cDNA with microarray chip and detect fluorescence.
46
Q

In regards to microarrays, does a human or a robot spot the genes onto a slide?

A

Robot.

47
Q

In regards to microarrays, why is the mRNA collected from two different cell samples compared?

A

For a direct comparison of their relative levels of gene expression.

48
Q

In regards to microarrays, what is the mRNA samples converted into?

A

cDNA.

49
Q

In regards to microarrays, cDNA from one source (control) is labelled with Cy3? What colour does this present as?

A

Green.

50
Q

In regards to microarrays, cDNA test is labelled with Cy5. What colour does this present as?

A

Red.

51
Q

In regards to microarrays, what happens to the labelled samples?

A

They are mixed in order to hybridise to become a microarray.

52
Q

In regards to microarrays, after incubation is complete, what occurs to the array?

A

It is washed and fluorescently scanned.

53
Q

In regards to microarrays, what does a green spot indicate?

A

The expression of the gene is higher in the control sample.

54
Q

In regards to microarrays, what does a red spot indicate?

A

The gene in the test sample is expressed at a higher level than the corresponding gene in the control sample.

55
Q

In regards to microarrays, what does a yellow spot indicate?

A

Genes are expressed at equal levels in both samples.

56
Q

In regards to microarrays, what does a dark spot indicate?

A

Little or no expression in either sample of the gene whose fragment is located at that position in the array.

57
Q

In regards to microarrays, what alternative manner can data be represented as?

A

A heatmap.

58
Q

Explain how a heatmap works in regards to the grid layout (rows and columns).

A

Each row represents a different gene and each column corresponds to different samples.

59
Q

In regards to a heatmap, areas that are predominately green represent what?

A

Down regulated genes in multiple samples.

60
Q

In regards to a heatmap, areas that are predominately red represent what?

A

Up regulated genes in multiple samples.

61
Q

In regards to a heatmap, areas that are black represent what?

A

Genes whose expression is not changed between samples.

62
Q

In regards to a heatmap, shade variations demonstrate what?

A

The degree of up or down regulation between samples.

63
Q

Define cluster analysis in regards to genetics.

A

Identify sets of genes that are coordinately regulated.

64
Q

State three advantages of sequencing the transcriptiome.

A
  1. Development of next generation sequencing technologies.
  2. Enable high throughput sequencing of multiple molecules.
  3. Unbiased.
65
Q

State four functions of single cell RNA-sequencing.

A
  1. Isolate single cells.
  2. Isolate mRNA.
  3. cDNA synthesis.
  4. RNA sequence.
66
Q

Name four methods to isolate single cells.

A
  1. Fluorescence-activated cell sorting.
  2. Optofluid-based cell handling.
  3. Microfluidic-based cell handling.
  4. Laser-capture microdissection.
67
Q

State three advantages of single cell RNA-sequencing.

A
  1. No issue with heterogeneity in cell types from tissue.
  2. Individual cancer cells from a tumour (no contaminating cells).
  3. Inter-cell variation in transcriptiome.

Antibody And DNA Technology (5 decks)

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