Product Class: Kit

p19 miRNA Detection Kit

Catalog #SizeConcentration
E3312S100 assays

Description

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:
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.
(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.

Features

Rapid detection of miRNA requiring few steps

Kit Components

  • p19 Binding Buffer
  • p19 Elution Buffer
  • BSA Treated Chitin Magnetic Beads (600 μl)
  • RNase Inhibitor, Murine
  • p19 siRNA Binding Protein
  • BSA
  • 1X p19 Wash Buffer

Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
p19 Binding Buffer10X
p19 Elution Buffer1X
BSA Treated Chitin Magnetic Beads (600 μl)
RNase Inhibitor, Murine-2040,000 units/ml
p19 siRNA Binding Protein-2010,000 units/ml
BSA-20100X
1X p19 Wash Buffer25X

Advantages and Features

Applications

  • miRNA detection without doing a Northern blot
  • Affinity purification of siRNA

Properties and Usage

Storage Temperature

-20°C

Supporting Documents

Material Safety Datasheets

The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

Notes

  1. Extensive washing is critical to reduce the background (non-specific binding of ssRNA probe to the chitin magnetic beads). We recommend the use of prewarmed (37°C) wash buffer and shaking of the beads during the incubation. Additional washing may be needed to reduce background.
  2. The small amount of p19 beads used for miRNA detection require careful pipetting. Care in the removal of the supernatent is necessary to avoid loss of the beads.

References

  1. Silhavy, D. et al. (2002). EMBO J.. 21, 3070-3080.
  2. Vargason, J.M. et al. (2003). Cell. 115, 799-811.
  3. Jin, J. et al (2010). BioTechniques. 48, XVII-XVIII.
  1. miRNA Detection (E3312)