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    Cat. No.
    Size
    List Price*
    QTY
    BIO-35028
    500 Reactions
    $580.00
    +
    BIO-35029
    2000 Reactions
    $1,670.00
    +
    *To check your pricing please Login or contact us

    Description

    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. Bioline has developed the DNA Extraction Control (DEC), which more closely mimics the test sample, as compared to spike controls. Genetic material from the test sample and the DEC 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 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 DEC 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. DEC 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
    Main
    Spike-in control and probe-based detection assay for simple validation of DNA extraction and determination of qPCR assay inhibition.

    Application Note

    DNA Extraction Control 670

    Instrument Compatibility

    DNA Extraction Control 670 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®.

    DNA Extraction Control 670 uses Quasar®670 and is also available with Cal Fluor® Orange 560 or Cal Fluor® Red 610, to fit in with existing protocols. CAL Fluor and Quasar dyes are performance-optimized fluorophores for multiplex qPCR.



    Specification

    Components

    Reagent

    500 Reactions

    2000 Reactions

    Internal Control DNA*

    5 x 500 µL

    20 x 500 µL

    25x Control Mix (containing Quasar® 670 labeled probe)

    5 x 100 µL

    20 x 100 µL


     * The Internal Control DNA is in viable α-Select 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 kit components should be stored at -20°C upon receipt. When stored under the recommended conditions and handled correctly, full activity of the kit is retained until the expiry date on the outer box label.

    Shipping conditions

    Shipped on dry/blue ice.



    FAQs

    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.