Preparation of the Probes

Fluorescent probes must be prepared, and these hybridise to the microarray. These are prepared from messenger RNA from the cells or tissues of interest.
Extraction of the mRNA is made easier by the identification of a key property by which the mRNA can be isolated.
See Introduction

Messenger RNA

Messenger RNA is produced from the DNA template of each gene, and signals specific protein production. This is the process of gene expression, and gene expression levels are dictated physiologically by a combination of genetic and environmental factors.
The genetic factors include gene regulatory regions such as promoters, enhancers and splice sites. The environmental factors can include temperature, light and stress.
It is the ease of obtaining fluorescent probes from mRNA of cells of interest, and also the ease by which these probes can be studied by hybridisation-based assays that makes mRNA so useful in functional genomics.

After the mRNA has been extracted from the cells or tissues under study, it is converted into DNA by the use of the reverse transcriptase enzyme.

During this reaction, the DNA is labelled by the incorporation of fluorescent or radioactive nucleotides into the DNA.
The two samples are labelled using two different fluorescent dyes - say, red or green.
This labelled DNA is then hybridised to the microarray slide.
Using mixtures that are differentially labelled allows the ratio of fluorescence to be measured, therefore avoiding most of the problems of hybridisation kinetics.

Fluorescent Dyes

Cy3 and Cy5 are presently the best two dyes for use in cDNA arrays. This is due to their:
  • widely separated absorption and emission spectra,
  • high molar absorption coefficients, and
  • good fluorescence quantum yields.

However...

Whilst the direct labelling of the DNA with a fluorescent group is the most simple method, it can also be problematic. The DNA is often short and varied in sequence. This means that finding one hybridisation condition that is optimal for every DNA spot is not possible. However it is generally possible to find saline and temperature conditions that give the desired strong signals for the hybridisation products, and weaker signals for the mismatches.


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