miRNA Detection (E3312)
miRNA Probe Design: Most miRNAs are 19 to 25 bases in length. To detect miRNA using p19 beads, the optimum size RNA probe is 19 nucleotides long and complementary to a target miRNA. The probe should form a blunt ended duplex with one end of the miRNA, however, the miRNA can extend several nucleotides past the other end of the RNA probe and still bind tightly to the p19 beads. The RNA probe should not have a 5´ phosphate so it can be labeled (see following section).
- Labeling of miRNA Probe: Combine the following in a sterile microcentrifuge tube:
- 20–100 ng 5´ OH RNA oligo
- 2 μl T4 Polynucleotide Kinase Buffer (10X)
- 20–100 μCi of γ-32P-ATP with 6,000 Ci/mM
- 2 μl T4 Polynucleotide Kinase (10 units/µl) (NEB #M0201)
- Add H2O to 20 μl
- Incubate at 37°C for 60 minutes followed by 20 minutes at 65°C to inactivate the enzyme. Use CentriSep column (Princeton Separation) to remove the unincorporated isotope and measure the specific activity using a scintillation counter.
- Standard Curve: For quantitative detection of miRNAs, a standard curve is made using decreasing amounts of a synthetic miRNA (from 300 to 1 pg) and a constant amount of the 32P labeled probe (~500 pgm). The hybridization can contain several micrograms of total RNA, like yeast or bacteria, which does not contain the miRNA of interest. This will mimic miRNA detection in the unknown samples. The counts bound and eluted from the p19 beads can be plotted against the pg of the miRNA in the reaction. The steps for hybridization and processing the beads are described below (see Figure 2 on the product page).
- Hybridization: Typically, 1–0.5 ng of 32P-probe was hybridized to a total RNA sample (from 1–10 μg) by incubating at 55°C–65°C (depends on the Tm of your probe) for 2 hours in 10 µl containing 1X p19 Binding Buffer. Temperature depends upon base composition of the RNA. Hybridization allows the probe to form duplex with the endogenous target miRNA. We recommend the use of a PCR machine to avoid evaporation.
- Binding of p19 to BSA Treated Chitin Magnetic Beads: Resuspend the BSA treated chitin magnetic beads with a brief vortex and place 10 µl of the suspension into a 1.5 ml microfuge tube. Add 3 µl of p19 protein to the 10 µl beads suspension and incubate it on a bench top shaker, such as Orbits compact microtube shaker or an equivalent device for 10 –20 minutes at room temperature.
- Binding of the Hybrid miRNA: RNA probe to the p19 beads: Prepare RNA Binding Buffer by adding the following into a 1.5 ml microfuge tube: 6 µl RNase free H2O, 1 µl of 10X p19 Binding Buffer, 2 µl of 10X BSA (dilute from the 100X stock) and 1 µl of RNase inhibitor. After a brief mix, add the 10 µl RNA Binding Buffer to the RNA hybridization reaction to bring the final volume to 20 µl. Remove the supernatant from p19 chitin beads (from step 5) with a Magnetic Separation Rack. Add the entire 20 µl hybridization mix to resuspend the beads pellet. Incubate the RNA binding reaction by shaking for 1.5 hours at room temperature.
- Removal of Unbound RNA: Excess probe and unbound RNA are removed by washing the p19 beads with BSA-wash buffer. BSA-wash buffer is prepared from the 25X wash buffer and 100X BSA stocks by dilution with sterile water to a final concentration of 1X wash buffer and 1X BSA. Heat up the 1X BSA-wash buffer to 37°C. Remove supernatant from RNA-p19 beads binding reaction using the Magnetic Separation Rack. Wash the beads pellet in 500 µl of 1X BSA-wash buffer and shake on a bench top shaker for 5 minutes at room temperature. Repeat the wash five times. To minimize any loss of the beads during the wash steps, allow the beads to settle to the bottom of the tube before using the magnetic rack. Beads are drawn to the side of the tube using the magnetic rack and the supernatant is carefully removed with a micropipetor.
- Elution: After the last wash, remove as much of the supernatant as possible without touching the beads pellet. Add 40 μl of pre-warmed 1X p19 elution buffer to the beads pellet and incubate for 10 minutes at room temperature with shaking followed by another 10 minute incubation at 37°C. Carefully remove the supernatant containing miRNA:RNA probe hybrid from the microfuge tube using the magnetic rack. 5 to 10 μl of eluate is usually sufficient for analysis on a liquid scintillation counter or PAGE using a 20 % TBE gel.