Flexible System for Evaluating Microarray Experiments
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 Stratagene developed the SpotReport™ array validation system* to
assess many of the experimental variables often encountered when
performing microarray analysis. The system allows you to track and
determine RNA quality, hybridization efficiency, and signal
linearity. It also allows you to evaluate array sensitivity, dye
bias, and array orientation. The flexible and easy-to-use format
includes lyophilized positive and negative controls that, once
resuspended in your favorite spotting buffer, are ready to
spot. |
What is the SpotReport™
array validation system?
The SpotReport™ array validation system is a group of controls that
allow you to validate parameters for your microarray experiment. The
system includes a carefully selected set of exogenous DNA controls,
corresponding RNA spikes, and a series of negative controls that allow you
to evaluate the printing quality, hybridization conditions, and
sensitivity of the assay, as well as test the efficiency of the DNA
labeling reaction and RNA quality. All of the controls are provided in a
ready-to-use format.
What is the SpotReport Arabidopsis array
validation system?
Stratagene selected specific genes from A. thaliana involved in
plant-specific processes. These plant genes were evaluated for homology to
human, mouse, rat, and yeast sequences using public databases. No
significant homologies were found. Additionally, these sequences were
radiolabeled and hybridized to membranes containing cDNA libraries
representing 29 different human tissues to further demonstrate that there
is no cross hybridization to human sequences. This set of sequences can be
used as exogenous controls for several eukaryotic systems including yeast,
mouse, rat, and human. These sequences are available as either PCR
products or oligos (70-mers) for spotting onto arrays.
What is the SpotReport™ Alien™ array validation
system**?
As an evolution to the SpotReport system, Stratagene created 10
synthetic sequences that have no homology to any known nucleic acid
sequence. This lack of homology allows these Alien sequences to be used as
exogenous controls in any array system including yeast, mouse, rat, human,
bacteria, and plant. These sequences are available as either PCR products
or oligos (70-mers) for spotting onto arrays.
What are the advantages of using the SpotReport
Alien system?
The main advantage is that the Alien controls can be used with both
eukaryotic and prokaryotic arrays. In addition, these sequences have
similar GC content (approximately 50%) and low secondary structure. This
allows for efficient and even hybridization kinetics between the labeled
and spotted Alien DNA.
What is the difference between the SpotReport-3
and SpotReport-10 array validation systems?
SpotReport-3 includes three A. thaliana mRNA spikes and
corresponding ready-to-spot PCR products. SpotReport-10 includes 10 A.
thaliana mRNA spikes and the corresponding ready-to-use PCR products.
Both kits contain human ß-actin PCR product, human COT-1® DNA, 3x SSC
buffer, poly (dA)40-60 oligonucleotide, and salmon sperm
DNA.
Why are negative controls
included?
The negative controls are used to evaluate the hybridization
specificity and array printing quality of your microarray system. For
example, there should be no signal from 3x SSC spots. If signal is
observed, there may be incomplete pin washing during the printing process
or non-specific hybridization. The polyA, COT-1®, and salmon sperm DNA
verify that the poly T in the labeled cDNA target, the repetitive elements
within the labeled cDNA, and non-specific hybridization events have all
been effectively blocked during hybridization. If signal is observed from
these spots, the hybridization conditions may need to be
optimized.
Why is there a difference in the amount of
poly(dA)40-60, salmon sperm DNA, and human COT-1® DNA provided
in the kit compared to the amount of Arabidopsis PCR
products?
Smaller quantities of poly(dA)40-60, salmon sperm DNA, and
human COT-1® DNA are
provided because they serve as negative controls
and do not need to be printed on as many array locations as compared to
the Arabidopsis PCR products. A sufficient amount of
Arabidopsis PCR products is provided to allow for statistical
analysis of array results. Typically, statistical analysis is not
necessary for negative controls.
What primers are used to amplify the PCR
products in the SpotReport Arabidopsis systems? Are the GenBank accession
numbers for those genes available?
Gene-specific primers are used to PCR amplify each Arabidopsis gene.
The PCR product size and GenBank accession number for each gene are listed
in the instruction manual.
How much of the PCR products should be printed
on a microarray slide?
The amount of PCR products spotted depends on the type of arrayer and
the array chemistry. The same amount of DNA should be spotted for the
control PCR products and test PCR products. Based on Stratagene’s internal
testing, the amount of each PCR product provided with the SpotReport array
validation system is sufficient to print 1,000 microarray slides if each
PCR product is spotted 10 times per slide.
How much Arabidopsis thaliana or Alien
mRNA should be spiked for labeling?
The amount of mRNA to be added to your fluorescence-labeling reaction
is dependent upon the sensitivity of your assay. A good starting point is
to add 5 ng, 0.5 ng, and 0.05 ng of three different mRNA species to 100 mg
of the test and/or reference total RNA. If less than 100 mg of total RNA
is labeled, increase or decrease the amount of mRNA added to the labeling
reaction to maintain the ratio of mRNA to total RNA. The amount of mRNA
added to the labeling reaction can be increased or decreased as
needed.
Do the PCR products or the mRNA have a polyA
tail?
The mRNA spikes have a polyA tail, however the PCR products do not.
CY™ is a trademark of Amersham Biosciences Limited
* license reference 2, ** license reference 5
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