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    Cat No. Size List Price*
    BIO-66045 4 x 100 Reactions
    £445.00

    *Specify individual assay(s) by clicking on 'Select Assays' button. A 50% surcharge will be applied to individual assays (1 x 100 reactions) that are Designed & Validated upon order, this will appear in the shopping cart.

    

    The EPIK miRNA Select Assay Kit contains any four individual EPIK miRNA RT-qPCR assays (100 reactions per assay). Choose from between 1 and 4 different assays per kit. Assays that are already designed but need to be validated upon order will require an additional two weeks delivery time and assays that need to be both designed and validated upon order will require an additional three weeks delivery time.

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    Description

    The unique design of the EPIK™ miRNA Assays confer distinct advantages over other approaches to miRNA detection. A stem-loop structure-based assays are designed using proprietary thermodynamics-based algorithms, enabling EPIK™ miRNA Assays to discrimination between miRNA sequences with high similarity and SensiSMART™ qPCR technology leads to remarkable sensitivities as well as extremely low background, enabling the accurate detection of very low miRNA levels.

    Product Description

    Using advanced design concepts, a proprietary algorithm has been developed by MiRXES™ to create modified stem-loop miRNA specific reverse transcription primers and hemi-nested real-time PCR primer combinations, to maximize miRNA detection sensitivity while minimizing non-specific interactions. The assays use SYBR® Green rather than a probe-based system for detection, allowing rapid amplification when required (Fig. 1). The resulting real-time PCR assays use SensiSMART™ enabling detection of extremely low levels of miRNA with high specificity, allowing the discrimination between closely related miRNA sequences (Fig. 2).

    Performance of these assays have been validated to detect as few as 100 copies of template per RT reaction (Fig. 3) with excellent assay efficiency and linearity.

    The EPIK miRNA Lo-ROX Kit is designed to allow you to do 4 x 100 reactions from a choice of over 27,000 miRNA primer sets. You can choose from 1 up to 4 different miRNA primer sets.

    Main

    Introduction to EPIK Panels

    Overview, features and benefits of the EPIK Panels

    EPIK™ miRNA Panel and Select Assay Workflow

    A summary of how to perform the assay protocol.

    The experimental procedure of the EPIK™ miRNA Select Kit is very easy. Multiplex cDNA synthesis for 3 miRNA targets and 1 reference target is highly efficient and led to very stable and reproducible results in QRT-PCR.

    Professor Dr. Peter Bugert, Institut für Transfusionsmedizin und Immunologie Mannheim, Germany

    Instrument Compatibility

    ABI 7500, 7500 FAST, ViiA7™, Stratagene (Agilent) Mx4000™, Mx3000P™, Mx3005P™, QuantStudio™ 3, 5, 6 and 12, as well as other instruments that do not require the use of a passive reference.


    Resources

    Resources

    Certification of Analysis (COAs)

    Specification

    Components

    20x RT Primers 4 x 16 µl
    10x PCR Primers 4 x 200 µl
    EPIK RT Buffer 1 x 160 µl
    EPIK RT Enzyme 1 x 40 µl
    2x SensiSMART™ Lo-ROX Master Mix 4 x 1 ml
    DEPC Water 5 x 1.8 ml

    Storage & Stability

    All components are shipped on dry/blue ice and should be stored at -20°C upon receipt for optimum stability. Repeated freeze/thaw cycles should be avoided. When stored under the recommended conditions and handled correctly, full activity of the reagents is retained until the expiry date on the outer box label.

    Shipping conditions

    On Dry Ice or Blue Ice.



    FAQs

    Yes the EPIK miRNA Panels and Select Assays can be are designed and validated to be used on any samples from the organisms and miRNAs listed in the mirBASE (please contact Customer Support for further information).

    No, EPIK miRNA assays are designed to measure exclusively the expression of mature miRNA, not pre-miRNA.

    No, the primers for the EPIK Panel Assays are pooled, so that one total RNA sample makes all of the different cDNAs for each of the plates. The EPIK Select Assays include individual RT-primer for every target miRNA, but we recommend to pool the primer for cDNA synthesis.

    Yes, the use of three miRNA specific primers means that EPIK can distinguish miRNAs that differ by only one single nucleotide.

    Other stem loop primers are several bases that correspond to the miRNA and the remainder of the primer is the same (universal). The universal sequence is used by the reverse nested qPCR primer, so that only the forward qPCR primer is specific. EPIK Stem loops are also several bases that correspond to the miRNA, however the remainder of the primer sequence is also unique. This means that two miRNA specific qPCR primers are used for the qPCR assay.

    Ensure your RNA samples have been prepared by a method that preserves low molecular weight RNAs, such as the ISOLATE II biofluids kit or the ISOLATE II miRNA kits.

    Yes, total RNA is the recommended starting material for the EPIK miRNA system.

    Phenol-based purification leads to bias in the miRNA and so is not recommended for the extraction of total RNA.

    This will depend on the expression level of the miRNAs, 10 pg of total RNA is sufficient for accurate quantification of highly expressed targets, whereas up to 100 ng may be required for low expression miRNAs.

    Either purified miRNA or total RNA can be used. If purified miRNA is used however, we recommended that the ISOLATE II miRNA Kits are used for the preparation of the samples, as this allows rapid, unbiased, phenol-free isolation of miRNA.

    Try less input RNA especially if the higher end of the recommended range had been used previously. Remember to use the same volume of template in each reaction.

    We recommend normalizing the samples at the total RNA level. Although the ratio between total RNA and specific miRNA is not fixed, measurement of total RNA provides a convenient way of estimating miRNA loading and an approximate methods for normalizing between experiments.

    The EPIK Assays are sensitive enough to measure samples below the recommended concentration of 100 ng total RNA. As low as 10 pg of total RNA is sufficient for accurate quantification of highly expressed targets whereas up to 100 ng may be required for low expression miRNAs. Typically the assays detect as few as 100 copies of template per RT reaction with excellent assay efficiency and linearity.

    Currently we do offer primer for the detection of the most commonly used endogeneous controls U6 snRNA and 5S RNA, but it is also possible to choose another stably expressed miRNA as internal control. We recommend to have a look into the related literature to find out common control genes for your experimental conditions. The consistency of expression has to be reconfirmed under the specific conditions of each experiment.

    The EPIK miRNA Select Assays comprise a choice from a growing list of over 1200 carefully selected miRNAs listed in the current version of miRBase.The number of possible miRNA targets for these assays increases constantly and will be adapted to further changes in miRNA research. If you can’t find your miRNA of interest within this list, you can request the miRNA on the EPIK miRNA Select Assay web pages.

    Reviews

    "The experimental procedure of the EPIK™ miRNA Select Kit is very easy. Mu ..."