p19 miRNA Detection Kit

M0310 product is a suitable alternative for most applications.

  • This product was discontinued on 12/09/2015

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E3312
  • Discontinued
    Discontinued
  • Product Information
    The p19 miRNA Detection Kit uses the high affinity binding to siRNA of the p19 protein to detect miRNAs that form hybrids with a specific probe. The p19 protein (19 kDa) from Carnation Italian Ringspot Virus (CIRV) binds 21–23 mer dsRNAs with nanomolar affinity (1) in a size dependent and sequence independent manner. The MBP aids in p19 purification and the CBD allows p19 to bind very tightly to Chitin Magnetic Beads (NEB #E8036). The p19 fusion protein bound to chitin magnetic beads (p19 beads) has the same binding properties as the native p19; it selectively binds siRNAs that are 21–23 bases long but does not bind ssRNA or dsDNA of the same length (2,3). After siRNA or hybrids of miRNA:RNA-probe are bound to the p19 beads, they can easily be isolated in a small volume using a Magnetic Separation Rack (NEB #S1506 or #S1509). The p19 beads can also enrich siRNAs greater than 3,000 fold from a mixture of total cytoplasmic RNA. 

    The use of p19 for miRNA detection has the dual advantage of high affinity and size dependent binding of dsRNA. Hybridization of a labeled RNA probe to a specific miRNA creates a dsRNA hybrid that selectively binds to p19 chitin magnetic beads. The unbound RNA's and probe are removed by washing. The eluted double stranded miRNA:RNA-probe can then be quantitatively measured by PAGE or a scintillation counter.. Using a 32P radioactive RNA probe, less than 10 picograms of miRNA can be detected in a million fold excess of unlabeled RNA (Figure 1). 

    p19 Beads Capacity:
    10 µl of p19 Beads suspension is enough to bind 300 ng of ds miRNA:RNA-probe. 

    1X p19 Binding Buffer:
    20 mM Tris-HCl
    100 mM NaCl
    1 mM EDTA
    1 mM TCEP
    0.02% Tween-20
    (pH 7.0 @ 25°C) 

    1X p19 Wash Buffer:
    20 mM Tris-HCl
    100 mM NaCl
    1 mM EDTA
    (pH 7.0 at 25°C)
    supplement with 1X BSA (100 µg/ml) 

    1X p19 Elution Buffer:
    20 mM Tris-HCl
    100 mM NaCl
    1 mM EDTA
    0.5 % SDS
    (pH 7.0 at 25°C)

    Figure 1: Figure 1

    The miRNA-probe, shown in red, is hybridized with a total RNA extract and bound to the chitin magnetic beads. The p19 protein, in green, is linked to the beads via the chitin binding domain. The unbound probe is removed and the miRNA:RNA-probe can be eluted and quantitatively measured.
    Figure 2: Standard curve for miR-122a in rat liver RNA. Figure 2

    (A) An autoradiograph of a 20% polyacrylamide TBE gel was used for the miR-122a standard curve. Increasing amounts of synthetic miR-122a were hybridized to a constant amount of 32P labeled probe in the presence of a large excess of Jurkat cell RNA. The miR-122a:RNA probe hybrid was bound to the p19 beads, washed and eluted as described. The lane labeled C is the size standard for miR-122a:probe hybrid and probe. (B) Standard curve for miR-122a detection was based on an aliquot eluted from beads used for the gel in panel A. (C) Detection of miR-122a in different amounts of total rat liver RNA. Eluted miR-122a:probe hybrids were analyzed as described in panel A. The size standard for ssRNA probe is in lane C. (D) Results of quantitative measurement of miR-122a in total rat liver RNA. Three different amounts of total RNA were used. The standard curve in panel B was used to convert cpm to pg of miR-122a.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    1X p19 Wash Buffer 25 X
    p19 Elution Buffer 1 X
    Purified BSA -20 10 X
    BSA Treated Chitin Magnetic Beads (600 μl)
    p19 siRNA Binding Protein -20 10,000 units/ml
    RNase Inhibitor, Murine -20 40,000 units/ml
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