Measuring global mRNA expression

Question 1

Expression profiling

Answer 1

Measuring the expression of thousands of fines simultaneously to create a global picture of cellular function

Question 2

2 main techniques for measuring global mRNA expression

Answer 2

1. Microarrays

2. RNA sequencing

Question 3

DNA microarrays

Answer 3

A collection of microscopic DNA spots attached to a solid surface
Used to measure the expression levels of large numbers of genes simultaneously OR to genotype multiple regions of a genome
Good for identifying genes whose expression levels are changed in response to disease

Question 4

Each DNA spot…

Answer 4

…contains a picomolar amount of a specific DNA sequence (DNA probes) that can hybridise to cDNA
Hybridisation generally detected by fluorescence

Question 5

Process for DNA microarrays

Answer 5

1. mRNA extracted from cell and isolated
2. mRNA reverse transcribed into cDNA
3. cDNA ends labelled with a fluorescent tag (e.g. Cy3/Cy5)
4. Labelled cDNAs added to the glass slide, and will hybridise with any complementary DNA probes
   (a) Hybridisation generally carried out for 17h at 65 degrees
5. Slide washed to remove any cDNAs that have non-specifically bound to the microarray surface, so only strongly bound strands will remain hybridised
6. Slide scanned and fluorescence measured, which produces a grid - the strength of the signal from a spot depends on the amount of target cDNA that has bound to the DNA probes