Download DNA and RNA Extraction Controls Performance Summary

DNA and RNA Extraction
Controls
Performance Summary
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DNA Extraction Control (DEC) and
RNA Extraction Control (REC)
Simple: easy monitoring and validation of DNA extraction protocols
DEC or REC and sample added
Extraction
The DEC or REC consists of cells containing an internal control sequence
(with no known homology to any organism). These cells are spiked into the
target prior to lysis. Following extraction, control mix (primers and probes)
is added prior to amplification.
DEC or REC is added to the sample prior to lysis and nucleic acid extraction to assess the
effects of extraction as well as PCR inhibition throughout the entire workflow
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DNA Extraction Control (DEC)
Specific: minimal interference with sample detection
Amplification curve for target gene
Singleplex
Amplification curve for DNA Extraction Control
Singleplex
Duplex
Duplex
Bioline have specially developed a DNA Extraction Control (DEC), which
more closely mimics the test sample and so does not interfere with the
target gene during real-time PCR.
Adding the DEC to your sample will not interfere with the extraction process or the real-time
PCR and so it can be used on all samples
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DNA Extraction Control (DEC)
Confirms extraction: DEC is extracted in exactly the same was as the
sample
Amplification curve for target gene
Amplification curve for DNA Extraction Control
No lysis
buffer
Complete
Lysis
No lysis
buffer
No
binding
buffer
Complete
Lysis
No
binding
buffer
Inefficient DNA extraction was shown by either not adding lysis buffer to
the sample, or not adding binding buffer to the column during the
extraction process.
The change in Cq for the DEC is similar to the target when there is poor extraction. As the same
volume of DEC is added to the samples each time, (which will give the same Cq each time)
changes to the DEC Cq can be used to monitor the extraction process
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RNA Extraction Control (REC)
Confirms extraction: REC is extracted in exactly the same was as the
sample
Amplification curve for REC
Complete
Lysis
Amplification curve for spiked DNA
No lysis/
No lysis binding
buffer
No buffer
binding
buffer
No lysis
No lysis buffer
buffer
Complete
Complete
Lysis
Lysis
No lysis/
No
binding
Nobinding buffer
binding
buffer
buffer
Inefficient DNA extraction was shown by either not adding lysis buffer to
the sample, or not adding binding buffer to the column during the
extraction process.
The change in Cq for the DEC and REC is similar to the target when there is poor extraction,
however spiking in DNA makes no difference to the Cq value, so it cannot be used to determine
extraction efficiency
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DNA Extraction Control (DEC)
Shows inhibitors: DEC is also effected by inhibitors in exactly the same
was as the sample
Amplification curve for target gene
No EDTA
0.5mM EDTA
1mM EDTA
2mM EDTA
2.5mM EDTA
3mM EDTA
3.5mM EDTA
4mM EDTA
Amplification curve for DNA Extraction Control
No EDTA
0.5mM EDTA
1mM EDTA
2mM EDTA
2.5mM EDTA
3mM EDTA
3.5mM EDTA
4mM EDTA
Increasing levels of inhibition was shown by increasing the concentration
of EDTA to the sample after extraction.
The DEC not only serves as an indicator of the effectiveness of the extraction process, but can
also be used to monitor co-purification of PCR inhibitors, as the DEC exhibits a similar profile of
inhibition to the target gene
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RNA Extraction Control (REC)
Shows inhibitors: REC is also effected by inhibitors in exactly the same
was as the sample
Amplification curve for RNA Extraction Control
No EDTA
0.5mM EDTA
1mM EDTA
1.4mM EDTA
1.8mM EDTA
Increasing levels of inhibition was shown by increasing the concentration
of EDTA to the REC after extraction.
The REC not only serves as an indicator of the effectiveness of the extraction process, but can
also be used to monitor co-purification of PCR inhibitors
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Conclusion
A common practice in real-time PCR is to add a known amount of “spiked” control
DNA after nucleic acid extraction. This monitors PCR inhibition but has no value as
an extraction control. The ideal situation is to have the test sample and internal
control undergo the same processing prior to real-time PCR. Bioline have specially
developed a DNA Extraction Control (DEC) and RNA Extraction Control (REC),
which more closely mimic the test sample, as compared to spike controls. Genetic
material from the test sample and the DEC or REC is simultaneously extracted by
common extraction methods, with the extraction control being as sensitive to
inhibition and extraction failure as the test sample.
The DEC/REC cells are of a known concentration, containing the Internal Control
DNA or RNA sequence. This sequence contains no known homology to any
organism and, importantly, has minimal interference with detection of sample. The
DEC/REC cells are spiked into the lysis buffer with the target sample, prior to DNA
extraction. Control Mix (primers and probe) is then added to the reaction mix before
amplification. Signal derived from the Internal Control confirms the success of the
extraction step. DEC/REC also monitors co-purification of PCR inhibitors that may
cause biased or false amplification patterns.
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Thank you……
…for your attention
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Kits
Product
DNA Extraction Controls
DNA Extraction Control 560
DNA Extraction Control 560
DNA Extraction Control 610
DNA Extraction Control 610
DNA Extraction Control 670
DNA Extraction Control 670
RNA Extraction Controls
RNA Extraction Control 560
RNA Extraction Control 560
RNA Extraction Control 610
RNA Extraction Control 610
RNA Extraction Control 670
RNA Extraction Control 670
Size
Cat No.
500 reactions
2000 reactions
500 reactions
2000 reactions
500 reactions
2000 reactions
BIO-35031
BIO-35032
BIO-35033
BIO-35034
BIO-35028
BIO-35029
500 reactions
2000 reactions
500 reactions
2000 reactions
500 reactions
2000 reactions
BIO-35044
BIO-35045
BIO-35048
BIO-35049
BIO-35040
BIO-35041
The DEC and REC Extraction Controls use Quasar ® 670, Cal Fluor® Orange 560 or
Cal Fluor ® Red 610, to fit in with existing protocols in a multiplex real-time PCR.
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