KJB - Transcriptomics

Question 1

What is an -ome?

Answer 1

A suffix that is attached to biological entities for describing very large-scale data collection and analysis.

Question 2

Define proteome.

Answer 2

All of the proteins produced by a cell at a given point in time.

Question 3

What does transcriptomics and proteomics entail?

Answer 3

Involves looking at RNA and proteins produced by a given set of cells under a given set of conditions.

Question 4

What is a gene expression profile?

Answer 4

It looks at all of your genes (20,000 - 30,000) at the same times and tells you which are on and off. If they are on, they will produce RNA + protein. If they are switched off, they will not. You can tell how much a gene is switched on by how much RNA and how many proteins are made. Some genes need to produce more product depending on what they are used for.

Question 5

Why use gene expression profiling and how is it done?

Answer 5

To help characterise complex diseases.

1. Collect tissue samples from healthy and diseased participants.
2. Determine gene expression profiles.
3. Identify genes that are expressed differently in.
4. Use this information to:
   
   • develop diagnostic tests
   • identify new drug targets

Question 6

What is a transcript?

Answer 6

An RNA molecule?

Question 7

Which is bigger, RNA or DNA?

Answer 7

DNA

Question 8

Which is more water soluble, RNA or DNA?

Answer 8

RNA

Question 9

Which is very unstable and degrades quickly, RNA or DNA?

Answer 9

RNA

Question 10

What techniques allow you to identify new drug targets?

Answer 10

Microarray gene chips, Spotted arrays and Affymetrix gene chips.

Question 11

What is the basic principle of micro array gene chips?

Answer 11

Nucleic acid hybridisation

Question 12

Microarrays are a solid support which contain…

Answer 12

… short pieces of single stranded DNA (probes) attached to it.

Question 13

A single strand of DNA or RNA can pair specifically with a second strand of complementary nucleotide sequence via which bond?

Answer 13

Hydrogen bond

Question 14

The probes are…? What is added for it to pair?

Answer 14

Single stranded DNA. We add RNA sequence for it to pair.

Question 15

Spotted arrays are also known as…

Answer 15

… two channel microarrays

Question 16

The conditions to make comparisons between healthy and diseased samples using spotted arrays are:

Answer 16

1. You must let them stick to the array at the same time

2. You can only make comparisons between two samples on the SAME chip.

Question 17

Do spotted arrays look at the whole set of genes?

Answer 17

NO. Only looks at a limited number of genes.

Question 18

How are Affymetrix gene chips synthesised?

Answer 18

Photolithographically. Robots synthesis probes directly onto the glass support of the chip.

Question 19

Difference between comparison making with spotted arrays and affymetrix gene chips?

Answer 19

Spotted arrays - can only make comparisons on the SAME chip.

Affymetrix gene chips - can make comparisons BETWEEN chips.

Question 20

Difference in how Affymetrix gene chips (Photolithographic oligo chips) and spotted arrays express intensity.

Answer 20

Affymetrix chips - BRIGHTER chips are more strongly expressed.
Spotted arrays - BIGGER chips are more strongly expressed.

Question 21

What are the 5 current applications of microarray technology?

Answer 21

1. Identifying new drug targets
2. Testing cellular activity of new drug molecules
3. Establishing toxicity profiles and identifying metabolic pathways of new drugs
4. Improving/ early diagnosis of conditions
5. Matching optimised drug treatment regimes to patient/tumour genotypes (pharmacogenomics)

Question 22

Briefly describe the 5 current applications of microarray techniques.

Answer 22

1. Identify new drug targets for specific disease conditions.
   Pair-wise comparisons between healthy and diseased tissues allows us to examine the gene expression profiles. Any alterations during disease can indicate a pathway that results in disease. These can make useful drug targets.
2. Testing cellular activity of new drug molecules.
   Cell cultures are treated with the novel drug molecule and the genome response is monitored. This also allows us to examine specificity/ pharmacology –> metabolism (including induction/inhibition)
3. Establishing toxicity profiles + identifying metabolic pathways of new drugs.
   Cell cultures and animal models are exposed to the new drug molecule. Existing gene expression profiles are compared with those that show signs of toxicity such as increase in inflammatory markers/ cell death.
4. Improved/ early diagnosis of disease condition.
5. Matching the optimum drug treatment to patient/ tumour genotype (pharmacogenomics).
   If a patient has poor prognosis, they are treated more aggressively. Gene expression profiles will allow us to choose treatment appropriately.