Expression Profiling

Question 1

Define transcriptiome.

Answer 1

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

Question 2

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

Answer 2

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

Question 3

What are the three methods of single gene analysis?

Answer 3

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

Question 4

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

Answer 4

1. Expressed sequence tags.
2. Microarrays.
3. Sequencing.

Question 5

State the purpose of Northern blotting.

Answer 5

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

Question 6

In Northern blotting, how are RNA samples denatured?

Answer 6

At 90°C and using formamide.

Question 7

In Northern blotting, how are RNA samples separated?

Answer 7

By size electrophoresis in a denaturing agarose gel.

Question 8

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

Answer 8

Formaldehyde.

Question 9

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

Answer 9

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

Question 10

In Northern blotting, how is mature mRNA isolated?

Answer 10

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

Question 11

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

Answer 11

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

Question 12

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

Answer 12

Sequences with partial homology.

Question 13

What four types of information can Northern blotting deduce?

Answer 13

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

Question 14

State the purpose of RT-PCR.

Answer 14

Amplify specific mRNAs by means of gene specific primers.

Question 15

What three pieces of information does RT-PCR provide?

Answer 15

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

Question 16

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

Answer 16

Complementary DNA (cDNA) transcripts from RNA templates.

Question 17

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

Answer 17

1. Random.
2. Oligo (dT).
3. Sequence specific.

Question 18

In PT-PCR, how is the locus amplified?

Answer 18

By means of specific primers.

Question 19

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

Answer 19

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

Question 20

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

Answer 20

Poisson.

Question 21

In digital RT-PCR, what does 0 represent?

Answer 21

A negative result.

Question 22

In digital RT-PCR, what does 0 represent?

Answer 22

A positive result.

Question 23

What is the last step of digital RT-PCR?

Answer 23

Absolute quantitation of number of copies of target.

Question 24

How are tissues prepared for in Situ hybridisation?

Answer 24

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

Question 25

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

Answer 25

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

Question 26

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

Answer 26

To hybridise intact tissues or embryos.

Question 27

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

Answer 27

UTP-digoxigenin.

Question 28

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

Answer 28

Proteinase K.

Question 29

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

Answer 29

mRNA.

Question 30

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

Answer 30

It is washed.

Question 31

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

Answer 31

Alkaline phosphate-conjugated antibody.

Question 32

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

Answer 32

Digoxigenin.

Question 33

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

Answer 33

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

Question 34

Define differential display.

Answer 34

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

Question 35

What two methods does differential display entail?

Answer 35

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

Question 36

State an advantage of using differential display.

Answer 36

There is no need for previous information about expressed genes.

Question 37

Define ESTs.

Answer 37

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

Question 38

How many nucleotides long are ESTs?

Answer 38

200 to 500.

Question 39

What are two advantages of ESTs?

Answer 39

Random and unbiased.

Question 40

ESTs can reconstruct complete sequences of what?

Answer 40

cDNA.

Question 41

What four pieces of information can be derived from ESTs?

Answer 41

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.

Question 42

What does dbEST (Genbank) stand for?

Answer 42

Database of EST sequences.

Question 43

What is the purpose of Unigene within Genbank?

Answer 43

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

Question 44

What are the two types of DNA chips?

Answer 44

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

Question 45

State the four steps of microarrays.

Answer 45

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.

Question 46

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

Answer 46

Robot.

Question 47

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

Answer 47

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

Question 48

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

Answer 48

cDNA.

Question 49

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

Answer 49

Green.

Question 50

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

Answer 50

Red.

Question 51

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

Answer 51

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

Question 52

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

Answer 52

It is washed and fluorescently scanned.

Question 53

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

Answer 53

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

Question 54

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

Answer 54

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

Question 55

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

Answer 55

Genes are expressed at equal levels in both samples.

Question 56

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

Answer 56

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

Question 57

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

Answer 57

A heatmap.

Question 58

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

Answer 58

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

Question 59

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

Answer 59

Down regulated genes in multiple samples.

Question 60

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

Answer 60

Up regulated genes in multiple samples.

Question 61

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

Answer 61

Genes whose expression is not changed between samples.

Question 62

In regards to a heatmap, shade variations demonstrate what?

Answer 62

The degree of up or down regulation between samples.

Question 63

Define cluster analysis in regards to genetics.

Answer 63

Identify sets of genes that are coordinately regulated.

Question 64

State three advantages of sequencing the transcriptiome.

Answer 64

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

Question 65

State four functions of single cell RNA-sequencing.

Answer 65

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

Question 66

Name four methods to isolate single cells.

Answer 66

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

Question 67

State three advantages of single cell RNA-sequencing.

Answer 67

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.