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    Cat. No.
    Size
    List Price*
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    BIO-66037
    2 x 2 96 well plates 0.1Y
    $1,067.00
    +
    BIO-66039
    2 x 2 96 well plates 0.2Y
    $1,067.00
    +
    BIO-66042
    2 x 2 96 well plates 0.1X
    $1,067.00
    +
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    Description

    The unique priming of the EPIK™ miRNA Assays confers distinct advantages over other approaches to miRNA detection. EPIK miRNA Assays use modified stem-loop, miRNA-specific oligonucleotide mediated reverse transcription and hemi-nested real-time PCR, combined with SensiSMART™ and SYBR® Green, offering remarkable sensitivities as well as extremely low background, thereby enabling the accurate detection of very low input miRNA levels and accurate discrimination of even very closely related miRNA targets.

    Product Highlights

    • Increased sensitivity RT and qPCR steps optimized to drive highly efficient amplification from limiting amounts (≥10 pg) of total RNA
    • Improved specificity pre-designed, novel, miRNA-specific primers for superior discrimination of even very closely related targets
    • Faster protocol RNA to Ct in less than 2 hours for earlier results and increased throughput capacity
    • Reliable data 7-log linear dynamic range for accurate quantification of low- and high- expressed targets in even limited sample volumes
    • Convenience all necessary components included in the kit and formulated to minimize set-up time

    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, to produce complete systems for miRNA profiling of biofluids.

    The EPIK Biofluid miRNA Panel Assay is made up of the most commonly studied 176 miRNAs in circulating biofluids (see plates). A set of controls present on this assay along with spike RNA enables data analysis, assessment of reverse transcription performance and assessment of PCR performance. Circulating miRNAs are regarded as a promising new class of biomarkers and a novel means of extra-cellular communications.

    EPIK miRNA Panel Assays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

    Download Excel file for Data Analysis

    If you need single assays, please contact Bioline Technical Support for advice.

    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.

    Instrument Compatibility

    Plate 0.1Y = ABI Fast 96-Well Block (0.1 mL, low profile) which is compatible with ABI ViiA7; ABI 7500 FAST; QuantStudio™ 3 Real-Time PCR System FAST; QuantStudio™ 5 Real-Time PCR System FAST; QuantStudio™ 6 Real-Time PCR system FAST; QuantStudio™ 7 Real-Time PCR system FAST; QuantStudio™ 12K Flex Real-Time PCR system FAST.

    Plate 0.2Y = ABI 96-Well Block (0.2 mL) which is compatible with ABI ViiA7; ABI 7500; QuantStudio™ 3 Real-Time PCR System; QuantStudio™ 5 Real-Time PCR System; QuantStudio™ 6 Real-Time PCR system; QuantStudio™ 7 Real-Time PCR system; QuantStudio™ 12K Flex Real-Time PCR system.

    Plate 0.1X = ABI 96-Well Block (0.1 mL) which is compatible with BioRad® CFX96; Roche LightCycler® 480 (96 well block only).


    Resources

     

    Certification of Analysis (COAs)

    Specification

    Components

    Biofluid Assay Plates 2 x 2 96 well plates
    RT Primer Pool - lyophilized 2 tubes (A and B)
    RNA Spike - lyophilized 1 tube
    EPIK RT Buffer 32 µL
    EPIK RT Enzyme 8 µL
    2 x SensiSMART™ Master Mix 4 x 1 mL
    DEPC Water 3 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 and the RNA Spike stored at -80°C after reconstitution. 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

    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.

    There is no universal 96 well plate for all qPCR machines, so you need to choose the correct plate type for your instrument, this can be done using the 'Choose the Right Plate' button on each of the panel pages.

    The list of miRNAs is found at the bottom of each product page and is displayed out as panels and as a list.

    We offer 3 distinct panel assays covering the most important miRNA in cancer and stem cell research as well as circulating miRNA (biofluid assay). If you are not sure which panel fits best for your needs, we suggest to check the enlisted miRNA and compare with the relevant literature for your field of interest. Should the problems persist, please contact technical services for further assistance.

    Yes, however all the cDNA reactions must be treated identically, so if it is not possible to run all the plates within one day, all the cDNA reactions must be frozen at ‐20°C once created. This will ensure that all cDNA reactions are subjected to the same number of freeze-thaw cycles.



    There are 88 unique miRNAs, 2 RNA Spike controls (in duplicate) and 2 interplate calibrators (in duplicate), however each plate is sold in duplicate so that the user can either perform a preliminary analysis of the experimental variation in one sample or perform an initial screen against two conditions as a single experiment (e.g. comparison of normal versus disease state).

    No, currently we only sell 96 well plates, please enquire about customization.

    MiRXES have their own software to search the literature for citations with each of the miRNAs and key words for the different types of plates, it then ranks these along with the weighting of the publication, in order to determine which miRNAs will be added to the plates. The ones that make it onto the plate form >90% of those that have been cited and are spread out randomly across the 2 plates (for stem cell and biofluid) or 4 plates (for cancer). While 10% may sound like a significant number, the importance of those miRNAs not included in the panel are individually of low importance. Essentially, there is a very long tail of miRNAs, each of which features somewhere in the relevant literature, that are considered of low importance because when the publications are considered collectively, each miRNAs is cited very infrequently – as little as just once.

    No, EPIK has been optimized to work with the EPIK RT enzyme and SensiSMART, as we could not guarantee results using other reagents, we do not sell them separately.

    0.1Y ABI 96-Well Block (0.1 mL, low profile), 0.1X ABI 96-Well Block (0.1 mL) and 0.2Y ABI 96-Well Block (0.2 mL), you need to choose the correct plate type for your instrument, this can be done using the 'Choose the Right Plate' button on each of the panel pages.



    This will depend on how many plates you need, please enquire about customization.

    The EPIK miRNA Panels allow the simultaneous relative determination of hundreds of miRNA species, either in duplicate with a single sample, or as single-panel assays of two samples. Raw data can then be exported into the Excel spreadsheet provided on our EPIK miRNA web page. These assays are not suitable for analysis of the absolute number of miRNA molecules in the sample, this requires individual miRNA assays in larger quantities.

    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.

    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.