Universal Control System for Validating Microarray Data
- Assess RNA quality and hybridization consistency
- Measure signal linearity and assay sensitivity
- Normalize data for dye bias
Microarray experiments involve many steps—all with the potential
for problems. It is difficult to determine if inconsistent or poor results
are due to problems with the RNA sample, labeling protocol, array, or
hybridization protocol.
The new SpotReport™ Alien™ Array
Validation System* allows you to optimize and control each
step of your microarray experiment. Ten synthetic sequences have been
designed with no homology to any known nucleic acid sequence. These synthetic
Alien sequences and corresponding RNA spikes can be used with plant, mammalian,
or microbial microarrays.
A Complete System
It’s easy to use the SpotReport Alien system (Figure 1). Choose
between oligo or PCR products depending on the type of arrays you are
printing. We provide enough material for at least 10 printings with each
Alien control in 10 different subarrays on each array. When labeling your
RNA, add in appropriate RNA spikes at varying concentrations. We also
provide human COT-1®, SSC, and polyA as negative controls and human ß-actin
as a other standard controls.
Assess RNA and Hybridization Quality
The quality of your test and reference RNA can be assesed by hybridizing
fluorescence-labeled human ß-actin cDNA to the ß-actin positive
control spotted on the array. Uniform hybridization across the array is
indicated by consistent hybridization signals from replicate spots of
each Alien control gene. Hybridization specificity is detected by the
lack of hybridization signal to the SSC, human COT-1®, and polyA spotted
on the array.
Measure Signal Linearity and Assay Sensitivity
Assay sensitivity, signal linearity, and dynamic range are easily determined
with the control system. By adding varying amounts of the 10 Alien RNA
spikes into the labeling reaction, you can create a semi-quantitative
standard curve. Analysis of the standard curve allows you to assess signal
linearity and sensitivity of fluorescent detection on standard two-color
microarrays (Figure 2).
Dye-Bias and Normalization
It is well known that Cy3 and Cy5 fluorescent dyes (Amersham Biosciences),
commonly used to label cDNA, are incorporated at different levels in reverse
transcription reactions and have different quantum yields. This results
in a difference in the Cy3 and Cy5 fluorescence intensities even when
equal amounts of Cy3- and Cy5-labeled cDNA are present. The SpotReport
system addresses this problem by enabling you to measure these differences
and normalize your data. You can normalize your data by (1) determining
the ratios of the hybridization signal of equal amounts of the Cy3- and
Cy5-labeled Alien cDNA and (2) multiplying the values from test or reference
cDNA by these ratios.
CY™ is a trademark of Amersham Biosciences Limited
* Patent pending
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