application note
1.16 Promega_ReliaPrep™_Blood gDNA isolation
Introduction
Trinean introduces a new cDrop™ application suited for
the quantification (ng/µl) of DNA extracted from blood
samples using the Promega ReliaPrep™ DNA isolation kit
(ReliaPrep™ Large Volume HT gDNA Isolation System,
Promega).
App description
Trinean’s proprietary spectral content profiling is used to
discern the fraction of genomic DNA (gDNA) from the
measured UV/VIS spectrum, thereby distinguishing it
from other absorbing sample constituents such as RNA,
proteins or hemoglobin. Using the specific DNA profile
found, the gDNA concentration is determined.
For proper use of this application:
-
Always use pure water as blank(s).
Turn off the all background correction options at the
start of the measurement or in the tab ‘single well
display’ (See user note UN012).
Included reference profiles:
The profiling algorithms in this app will decompose the
measured UV/VIS spectrum (Figure 1 right, white curve)
and extract these specific profiles:
•Nucleic Acids (brown):
This profile is composed of a 42% GC content DNA profile
as well as profiles for intact and degraded RNA.
•DNA (green):
The molecule of interest. This profile is specific for DNA
with a GC content of 42%.
•Impurities
- RNA / nucleotides: total and degraded RNA
- EDTA
- Protein: Average of human protein spectra
- Hemoglobin/hematin: Hb +hematin
•Turbidity (grey):
This profile combines the intrinsic samples turbidity and
scattering (from bead contamination or others), and
takes into account potential instrument-specific optical
effects.
TRINEAN NV
Dulle-Grietlaan 17/3
B-9050 Gentbrugge
Figure 1: cDrop™ analysis of genomic DNA using the
Promega_ReliaPrep Blood gDNA app. cDrop™ screenshot
illustrating the app reference list (left) and one sample content
analysis analyzed with cDrop™ (right).
Figure 1 (left) depicts the reference spectra used by the
Promega_ReliaPrep Blood gDNA app. Figure 1 (right)
shows the results of spectral content profiling with this
app of a sample that was extracted from cell lines using
the Promega_ReliaPrep Blood gDNA kit.
The RRSE-value relates to the quality of the spectral
fitting of the selected cDrop™ app. This value gives an
indication of the unidentified fraction of the measured
UV/vis spectrum. When this value is too high (> 2.5%),
the analysis is not reliable due to the presence of an
unknown chemical or by a too low concentration of DNA.
In this case the DNA and RNA values will be grayed out
and the concentration of total Nucleic Acids will be
highlighted.
Results
Comparison to Picogreen
Samples were provided by two independent institutes
and prepared by gDNA extraction from blood by Promega
Reliaprep gDNA kit. These were measured in
quadruplicate on DropSense96® and analyzed by the
cDrop™ app for this kit. The A260 measurements and
DNA results were compared to a reference method for
quantification of dsDNA (Quant-iT™ PicoGreen® dsDNA
Assay Kit, Life Technologies). In Figure 2, picogreen values
(green bars) are compared to DNA measured with the
ReliaPrep blood gDNA isolation app (red bars) and the
cDrop Application Note _ Promega_ReliaPrep™ Blood gDNA app
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application note
classic A260 concentration determined by DropSense96
(grey bars). These results show a good correlation
between picogreen and both the A260 and cDrop™
values measured by the DropSense96.
600
Comparison to reference methods
Conclusion
The Promega_ReliaPrep Blood gDNA isolation app can be
used for the specific quantification of gDNA isolated from
blood using the ReliaPrep™ gDNA Blood kit.
Picogreen
Concentration (ng/µl)
500
cDrop gDNA
DS96 A260 concentration
400
300
200
100
0
842
843
845
850
856
874
876
878
884
892
Figure 2: Comparative data picogreen – cDrop™ application
Promega_ReliaPrep™_Blood_gDNA
Example export results.
When exporting your data from cDrop™, the software
provides the possibility to design your report, as
illustrated in Figure 3. You can select the analysis
information you want to include in your report table and
can sort them by moving a column up and down. The
resulting lay-out can be seen in the preview section.
Upon creating the desired output table, you can select
your preferred export format like excel, csv, pdf or text.
Rev 07/2015
www.trinean.com
Figure 3: Exporting data from cDrop™ to an Excel file.
TRINEAN NV
Dulle-Grietlaan 17/3
B-9050 Gentbrugge
cDrop Application Note _ Promega_ReliaPrep™ Blood gDNA app
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