FAQs - Frequently Asked Questions
Controls
Q. What is a spike control and what can it be used for?
A. Spike controls are probes designed to detect sequences that are not present in the genome of interest. We can then add a fixed amount of the complementary targets to the labelling reactions and they can can then be used to correct systematic biases.
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. Do FlyChip have the same or similar controls as Affymetrix?
A. All of our gene expression microarrays include spike controls. Our latest long oligonucleotide microarray, additionally includes degradation controls. The genome tile microarrays that we print have sheared genomic DNA. All microarrays include spotting buffer only spots. Our microarrays are therefore comparable to Affymetrix.
For more information: Services » Core Drosophila Microarrays
For more information: Services » Analysis » Downloads
Q. At what point are the spike transcripts included and what do they tell you?
A. The spike transcripts are added before labelling. This means that all spots that represent spikes should produce a signal in both channels. Differential dye incorporation rates between the channels and local variations in hybridisation efficiency can then be seen in the spike controls spots. These biases can then be corrected by normalisation.
For more information: Protocols » Gene Expression
For more information: Services » Analysis
Q. Do you use the spikes during the normalisation?
A. We do not currently use the spikes during normalisation because the algorithms are not fully developed. We instead use variance stabilising normalisation. The spike data is included in the raw data files that we send to all users.
For more information: Services » Analysis
For more information: Services » Analysis » Downloads » FC004 help file
For more information: Services » Analysis » Downloads » FL002 help file
Q. Is there any control for overall background? Or is the background worked out on a spot by spot basis?
A. The spotting buffer only and not printed spots are negative controls because they should have no signal after hybridisation. The raw data files include local spot background measurements. We do not use background subtraction, as it increases varience.
For more information: Services » Analysis » Downloads » FC004 help file
For more information: Services » Analysis » Downloads » FL002 help file
