DiscovArray™ Performance Specifications
Performance specifications of the DiscovArray Service reflect the robust Affymetrix
platform and the proficiency of Asuragen's scientists.
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Performance Specifications
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Metric
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Asuragen's method
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Recommended Input
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200 ng total RNA (no fractionation required)
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LOD1
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< 2.5 amol
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Dynamic Range1
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3 logs
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Reproducibility (R2)1,2
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0.998 (median CV < 3%)
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Dose-Response Linearity (R2)1
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0.95
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Relative Accuracy1,3
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0.96 ± 0.12
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Sample QC
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Incoming sample QC by Bioanalyzer, Spectrophotometer, and qRT-PCR.
If, for any reason, a qualified sample does not pass our process control specifications,
we will re-run the sample at no additional charge.
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Footnotes:
Limit of Detection (LOD), Reproducibility, Dynamic Range, Dose-Response Linearity,
and Relative Accuracy are all based on latin-square spike-in study in a complex
background of bladder total RNA processed according to the DiscovArray Service protocols.
Reproducibility reflects linear R^2 values between full-process technical replicates.
Relative accuracy describes the degree to which the system responds to linear changes
in the concentration of spike-in controls. The dose response slope measures the
accuracy of reported fold-change relative to the known fold-change of a serial dilution
series of miRNA spikes. A slope of 1.0 indicates perfect relative accuracy; slopes
KD of approximately 0.8 are typical of microarray-based platforms as they are known
to compress data.
Two process replicates are demonstrated here, illustrating the range of spikes
used in the Latin-square design (35 amol to 9 fmol). Note that the range defined
by the spikes brackets the biology relevant range of the bladder total RNA.
Linear Dynamic Range
Outstanding correlation with qRT-PCR, even from fixed (FFPE) tissues
Eight FFPE blocks corresponding to four samples of two different tissue types (A
and B) from eight different patients were purchased from Proteogenex (Proteogenex,
Inc.). All paraffin blocks were less than two years old. Total RNA was extracted
from each of these blocks by Asuragen’s FFPE RNA isolation services team.
10ug of total RNA isolated from each FFPE sample was analyzed with the Asuragen
DiscovArray Service.
A series of qRT-PCR reactions were performed using TaqMan* microRNA assays specific
for 6 different miRNAs* following manufacturer’s instructions and with an input
of 15ng total RNA per reaction. Real-time reactions were run on the 7900HT fast
realtime PCR system* and initial analysis of data was done using the SDS 2.3 software
included.
The following figures demonstrate the remarkable correlation between the qRT-PCR
data and the data from the DiscovArray service, not only in fold-change difference,
but also statistical significance (p-values as shown). These p-values demonstrate
the overall reproducibility of the DiscovArray process. The overall correlation
between the array and qRT-PCR data is 0.958.
In all figures, the array data shown are VSN normalized values and the TaqMan data
are ΔCTs.
Interested in Asuragen's other miRNA services? [Click here]
* (Applied Biosystems, Foster City, CA, USA)