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Description
Formerly DNA Extraction Control 560.
DNA Extraction Control not only enables users of a diagnostic qPCR assay to determine if there are inhibitors in the PCR assay, but also to validate the success of the extraction step, reducing the chance of obtaining a false negative result in the sample DNA.
Product Highlights
Simple - streamlined protocol for straightforward validation of DNA extraction and determination of qPCR assay inhibition
Sensitive - control assay identifies even small effects on DNA extraction and inhibition of amplification
Optimized - control DNA has a sequence with no known homology to any organism thereby avoiding detection of sample DNA
Specific - probe-based assay designed specifically for real-time PCR assays
Flexible - ideal for use with a wide range of sample types, including inhibitor-rich samples like blood, urine and sputum samples
Product Description
A common practice in qPCR is to add a known amount of spiked control DNA after DNA 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 qPCR. Meridian has developed the qPCR Extraction Control, which more closely mimics the test sample, as compared to spike controls. Genetic material from the test sample and the qPCR Extraction Control is simultaneously extracted by common extraction methods, with the extraction control being as sensitive to inhibition and extraction failure as the test sample.
The qPCR Extraction Control cells are of a known concentration, containing the Internal Control DNA sequence. This sequence contains no known homology to any organism and, importantly, has minimal interference with detection of sample DNA. The qPCR Extraction Control cells are spiked into the lysis buffer with the target sample, prior to DNA extraction. Control Mix, which includes primers and probe, is then added to the reaction mix before amplification. Signal derived from the Internal Control DNA confirms the success of the extraction step. qPCR Extraction Control also monitors co-purification of PCR inhibitors that may cause biased or false amplification patterns.
Applications
Pathogen detection
Cancer risk assessment
Gene expression analysis
Copy number variation (CNV) analysis
Genotyping
Viral loading
Fig. 1 Illustration of the extraction process
DEC assesses effects of extraction as well as inhibition throughout the entire workflow.
Fig 2 Minimal interference of DEC in sample detection.
A/ β 2 Microglobulin (b2MG) was amplified in triplicate from human genomic DNA in singleplex (green) and in duplex with Internal Control (blue). B/ The Internal Control was amplified in singleplex (red) and in duplex with b2MG (blue). The Cts show no difference between singleplex (b2MG - green, Internal Control - red) and duplex (blue) reaction assays in both target gene and Internal Control.
Fig. 3PCR reaction inhibition
A/ A fragment of the β2MG gene was amplified from genomic DNA and B/ the internal control sequence from the DEC. Increasing concentrations of EDTA were included in the reaction to simulate increasing concentrations of an inhibitor. The results illustrate that DEC gives the same pattern of inhibition as with the sample target, showing that inhibition of PCR reaction can be identified using DEC.
qPCR Extraction Control Orange is suitable for use with commercially available silica-membrane DNA extraction kits and CHELEX matrices and has been tested on a wide range of qPCR platforms including ABI-7500, LightCycler 480®, RotorGene-Q™, Mic and MX3005P®.
qPCR Extraction Control Orange uses Cal Fluor® Orange 560 and is also available with Quasar® 670 or Cal Fluor® Red 610, to fit in with existing protocols. CAL Fluor and Quasar dyes are performance-optimized fluorophores for multiplex qPCR.
25x Control Mix 560(containing Cal Fluor® Orange 560 labeled probe)
20 x 100 µL
* The Internal Control DNA is in viable E. coli cells (genotype: F- deoR endA1 recA1 relA1 gyrA96 hsdR17(rk-, mk+) supE44 thi-1 phoA Δ(lacZYA-argF)U169Ф80lacZΔ15λ–pBR322 (ranseqb1 AmpR)).
Storage & Stability
All components should be stored at -80°C upon receipt. When stored under the recommended conditions and handled correctly, full activity is retained until the expiry date on the outer box label.
We recommend to use the probe based detection methods for quantification of DNA and RNA extraction controls. Also, the REC kits have been designed for one-step RT-qPCR. But, it would be possible to use other PCR applications for detection and quantification of the extraction controls. Also possible are two-step PCR, SYBR® based or even endpoint PCR. If you would like to use REC with a separated reverse transcription it would be necessary to use the provided primer Control Mix also for reverse transcription reaction. If you are using SYBR® based detection methods, it is possible in singleplex only. Otherwise, the Ct value will be influenced by both the amplification of the target and extraction control.
We recommend to use a one-step RT-qPCR setup for detection of the RNA extraction control, but it would be possible to perform a two-step qPCR with separate reverse transcription. It would be necessary for this to use the provided primer control mix also for reverse transcription as hexamer or oligo dT would not give consistent results. The customer needs to optimize the amount of primer control-mix for reverse transcription.
The DNA and RNA extraction controls are suitable with any kinds of extraction method. It may be combined with e.g. phenol, column or magnetic bead based extraction methods and it is also suitable with automated extraction. The genetic material from the test sample and DEC or REC are simultaneously extracted with any common extraction method.
The benefit of the artificial DEC or REC cells in evaluation of the extraction process is the possibility to validate sample extraction process. Signal derived from the Internal Control confirms the success of the extraction step and monitors co-purification of PCR inhibitors that may cause biased or false amplification patterns. Internal control or spike in samples do not monitor the whole extraction process, especially not the lysis step. Also with quantification of internal genes it is not possible to distinguish for instance between low extraction efficiency and degraded samples.
