FAQs - Frequently Asked Questions
Labelling and hybridisation
Q. How do you perform the labelling reactions?
A. We perform direct labelling because we find this technique to be more robust. We can also perform indirect labelling.
For more information: Protocols » Gene expression » Direct Labelling
For more information: Protocols » Gene expression » Indirect Labelling
Q. Which Dyes do you use and what is the incorporation rate of these dyes?
A. We use Cy3-dCTP and Cy5-dCTP dyes for direct labelling. We use Cy3 and Cy5 monofunctional for indirect labelling. Incorporation rate varies according to labelling efficiency.
For more information: Protocols » Gene expression » Direct Labelling
For more information: Protocols » Gene expression » Indirect Labelling
For more information: Protocols » Gene expression » Dye Incorporation
Q. Do you label total RNA?
A. We only label the mRNA because we use oligodT primers. We do not, however, purify the mRNA from the total RNA mixture before performing the labelling reactions because we find that this is not necessary.
For more information: Protocols » Gene expression » Standard extraction
For more information: Protocols » Gene expression » Direct Labelling
For more information: Protocols » Gene expression » Indirect Labelling
Q. How much of your spike control mix do you add to your labelling reactions?
A. We add 200-500 pg of each spike to 50 µg of total RNA. We match the spike mixture concentration to each microarray format, to make certain that the spike control spot signals are comparable to the other spot signals. We aim to cover the full range of spot signals.
For more information: Protocols » Gene expression » Direct Labelling
For more information: Protocols » Gene expression » Indirect Labelling
Q. What it the advantage of using an automated hybridisation station compared to manual hybridisation?
A. Automated hybridisation helps to reduce spatial biases caused by local variations in hybridisation efficiency, as the labelled hybridisation mixture is agitated during hybridisation. Automation also helps to reduce batch variance.
For more information: Protocols » Hybridisation
For more information: Facility » Equipment » GeneTac Hybridisation Station
Q. Why do you use a manual wash for hybridisations against the oligo microarrays?
A. Our protocols are optimised for each microarray type and for some formats we have found that manual washes increase the target sequence discrimination.
For more information: Protocols » Hybridisation
For more information: Facility » Equipment » GeneTac Hybridisation Station
