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EPIK™ miRNA Select Lo-ROX Kit

Cat No. Size List Price*
BIO-66045 4 x 100 Reactions
$665.00

*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 designed, validated upon order will require an additional two weeks delivery time and assays designed and validated upon order will require an additional three weeks delivery time.

Please Search or Select Your Assay:

Accession Number Mature miRNA ID Assay Availability Hidden
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Accession Number Mature miRNA ID Assay Availability Quantity

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.

Features & Benefits

  • An extensive choice Over 27,000 miRNA assays from all organisms on the miRBase
  • Increased sensitivity RT and qPCR steps optimized to drive highly efficient amplification from limiting amounts (≥1pg) of input sample
  • Improved specificity pre-designed, novel, miRNA-specific primers for superior discrimination of even very closely related targets
  • Faster protocol RNA to Ct in just 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

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.

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. Each assay is evaluated using over 36 design parameters before being extensively wet-lab validated using both synthetic miRNA template and reference human RNA. 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 a list of individual miRNA assays that are ideal for validation and determination of miRNA function.

The EPIK miRNA Select Assays comprise a choice from over 27,000 miRNAs listed in the current version of miRBase. Each kit contains primers specific for up to four miRNAs along with reverse transcriptase and sufficient SensiSMART for 400 real-time PCR reactions. If you cannot find the miRNA that you are interested in, please inquire..

EPIK miRNAs assays are also available as panels for the Cancer, Stem Cell and Biofluid screening.

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


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.



Frequently asked questions

  • Can EPIK miRNA Panel Assays be used for other organisms?

      Although there is overlap between the miRNA found in the EPIK miRNA Panels and those found in other organisms, the EPIK miRNA Panels are designed to be used on human samples.
  • Can EPIK miRNA Panel Assays be used for pre-miRNA detection?

      No, EPIK miRNA assays are designed to measure exclusively the expression of mature miRNA, not pre-miRNA.
  • Do I need to make the cDNA for each of the miRNAs separately?

      No, the primers are pooled, so that one total RNA sample makes all of the different cDNAs for each of the plates.
  • Can EPIK miRNA Panel Assays detect single nucleotide difference between miRNAs?

      Yes, the use of three miRNA specific primers means that EPIK can distinguish miRNAs that differ by only one single nucleotide.
  • How do other stem loop RT primers different to EPIK’s stem loop RT primers?

      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 one of the nested primers, so that only one specific primer is used qPCR. 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 primers are used for the qPCR.
  • Can I run the plates separately?

      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.
  • How many replicates are on each plate?

      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 singlicate experiment (e.g. comparison of normal versus disease state).
  • Is it possible to have the panels in a 384-well format?

      No, currently we only sell 96 well plates, please enquire about customization.
  • How did you choose the specific miRNAs in each of the panels?

      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.
  • Can I just buy the plate and use my own reagents?

      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.
  • What do ‘X’ and ‘Y’ stand for in the product names?

      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.
  • How should I prepare my total RNA samples?

      Ensure your RNA samples have been prepared by a method that preserves low molecular weight RNAs, such as the ISOLATE II biofluids kit.
  • Can I use total RNA for the EPIK miRNA Panel Assays?

      Yes, total RNA is the recommended starting material for the EPIK miRNA system.
  • Can I use standard phenol-based purification for extraction of the total RNA?

      Phenol-based purification leads to bias in the miRNA and so is not recommended for the extraction of total RNA.
  • What is the minimum amount of total RNA required for miRNA?

      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.
  • Is there an option to customize a panel with the miRNA’s we are interested in?

      This will depend on how many plates you need, please enquire about customization.
  • The protocol indicates total RNA should be used as starting material. Will I obtain better results if miRNA is used?

      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.
  • What is the limit of detection with the EPIK miRNA Panel Assays?

      Typically the assays detect as few as 100 copies of template per RT reaction with excellent assay efficiency and linearity.
  • Why did the real-time PCR yield Ct values <12?

      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.
  • What quantification methods can be used with the EPIK miRNA Panel Assays?

      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.
  • When do I normalize my samples prior to labelling?

      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.



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