microarray analysis allows detection/ comparison of expression levels of multiple genes. what...

Microarray Analysis

Allows detection/ comparison of expression levels of multiple genes.

What molecule could be used as an indicator of expression?

DNA?

No! There are exactly two copies of every* gene in each cell.

RNA?

Perfect! The number of mRNA transcripts in a cell is proportional to expression level of the corresponding gene.

Well, almost perfect . . .

mRNA is much less stable than DNA

Solution:

•Isolate mRNA from tissue•Immediately reverse transcribe •Incorporate a fluorescent tag

Result= Fluorescently labeled cDNA

5’ 3’AAAAAA

TTTTTTTT Cy5Fluorescent Tag

5’ 3’AAAAAATTTTTTT

Isolate RNA from Source A (e.g., yeast cells grown under aerobic conditions)

Isolate RNA from Source B (e.g., yeast cells grown under anaerobic conditions)

Synthesize cDNAs against the mRNA templatesusing fluorescently labeled oligo dT primers.

Cy3Fluorescent Tag

Combine the two populations of

cDNAs

Apply to microarray

Oligonucleotide Synthesis

(How arrays are “spotted”)

Aerobic Respiration

Anaerobic Respiration

Gene A expressed?

yes yes

Gene B expressed?

yes yes

Gene C expressed?

yes no

Gene D expressed?

no yes

Gene E expressed?

yes yes

Compare expression profiles of

yeast grown under

anaerobic conditions and

yeast grown under aerobic

conditions.

C and D

Which gene(s) should be investigated for a potential role in anaerobic respiration?

Gene Result (Color)

C4BPA

ODC1

SIAT9

FGG

HBG1

CYP24

Our Microarray simulates expression levels of the six genes assigned for the Genome Scavenger Hunt

Red indicates expression in malignant lung tissue

Blue indicates expression in normal lung tissue

Color intensity is proportional to expression level.