Performance Specifications of Asuragen Labeling method combined with Affymetrix
GeneChip® miRNA Array*
Performance specifications of the miRNA Profiling Service using the Affymetrix GeneChip
miRNA Array reflect the robustness of the Affymetrix platform and the superiority
of Asuragen's labeling method.
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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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100 ng total RNA for tissues (no fractionation required); 200 ng for FFPE and cell
lines
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LOD1
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< 1 amol
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Dynamic Range1
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> 2.5 logs
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Reproducibility (R2)2
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0.97 (median CV < 4%)
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Dose-Response Linearity (R2)1
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0.99
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Relative Accuracy1,3
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1.02 ± 0.26
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Specificity4
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< 15% cross-hyb signal (average) from highly related, similar sequences with > 1
nt sequence differences
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Sample QC
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Incoming sample QC by Spectrophotometer, and TaqMan RT-qPCR.
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:
1... Limit of Detection (LOD, Dynamic Range, Dose-Response Linearity,
and Relative Accuracy are all based on latin-square spike-in study in a complex
background of placenta total RNA processed according to the Affymetrix GeneChip
Array Service protocols using the Asuragen labeling method.
2... Reproducibility reflects linear R2 values between full-process technical
replicates.
3... 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 of approximately 0.8 are typical of microarray-based platforms
as they are known to compress data.
4... Specificity of Affymetrix GeneChip Array is based on performance
assessment on let-7 family members.
Linear Dynamic Range
Two process replicates are demonstrated here, illustrating the range of spikes
used in the Latin-square design (1 amol to 316 amol). Note that the range defined
by the spikes brackets the biologically relevant range of the placenta total RNA.
Outstanding correlation with RT-qPCR and other commercially available platforms,
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 Inc (Culver
City, CA). 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. 100 ng
of total RNA isolated from each FFPE sample was analyzed using the Affymetrix GeneChip
miRNA array and Asuragen labeling method; 200 ng of total RNA was analyzed using
the Agilent V2 miRNA Array and 500 ng of total RNA was analyzed with the Asuragen
miRLink Service.
A series of qRT-PCR reactions were performed using TaqMan microRNA assays#
specific for 6 different miRNAs following manufacturer’s instructions and using
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 RT-qPCR
data and the data obtained from different array-based platforms with high statistical
significance (p-values as shown). These p-values demonstrate the overall reproducibility
of the Asuragen profiling service using the Affymetrix GeneChip miRNA array and
Asuragen labeling method.
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)
*...For Research Use Only. Not for use in diagnostic procedures.