In this protocol

适用说明:本说明书适用于KAPA RiboErase 试剂盒 (KK8481和KK8482)。
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Product Description

The KAPA RiboErase Kit (Human/Mouse/Rat or HMR) contains all of the buffers and enzymes required for depletion of ribosomal RNA (rRNA) from 100 ng – 1 μg of total RNA via the following steps:

  1.  hybridization of DNA oligonucleotides complementary to rRNA of human, mouse, and rat species;
  2. RNase H treatment to remove rRNA duplexed to DNA oligonucleotides; and
  3. DNase treatment to remove original DNA oligonucleotides.

The kit provides all of the enzymes and buffers required for rRNA depletion, but does not include RNA, RNase-free water, or beads for cleanup steps.

Product Applications

The KAPA RiboErase Kit (HMR) is designed for both manual and automated depletion of rRNA from 100 ng – 1 μg of total RNA. The kit depletes both cytoplasmic (5S, 5.8S, 18S, and 28S) and mitochondrial (12S and 16S) rRNA species.

Process Workflow

Ribosomal RNA Depletion Protocol

  1. Reagent Preparation
    For maximum stability and shelf-life, enzyme and reaction buffers are supplied separately in the KAPA RiboErase Kit (HMR).

    In the examples given in the following tables, all of the required water is included in each master mix, allowing the entire reaction mix to be added in a single pipetting step. Additional reagents required for the KAPA RiboErase protocol are listed inTable 4.

    Always ensure that KAPA Pure Beads are fully equilibrated to room temperature before use.

    Table 1. Oligo hybridization


    Table 2. rRNA depletion


    Table 3. DNase digestion


    Table 4. Volumes of additional reagents required


  2. Oligo Hybridization and rRNA Depletion 
    This protocol requires 100 ng – 1 μg of total RNA, in 10 μL of RNase-free water.

    Ensure that the hybridization master mix (Table 1) and the depletion master mix (Table 2) are prepared and kept at room temperature before use.
    2.1
    Program a thermocycler as follows:


    2.2
    Assemble rRNA hybridization reactions as follows:


    2.3
    Place samples in the pre-programmed thermocycler and execute the program.
    2.4
    Ensure the depletion master mix containing RNase H is added while the samples are kept at 45°C in a thermocycler. When the program reaches the pause step at 45°C, add the following to each 20 μL hybridization reaction.


    2.5
    Resume the cycling program to continue with the depletion step (45°C for 30 min).
    2.6
    Proceed immediately to rRNA Depletion Cleanup (Step 3).

  3.  rRNA Depletion Cleanup
    3.1
    Perform a 2.2X bead-based cleanup by combining the following:


    3.2
    Thoroughly resuspend the beads by pipetting up and down multiple times.
    3.3
    Incubate the plate/tube(s) at room temperature for 5 min to bind the RNA to the beads.
    3.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    3.5
    Carefully remove and discard 75 μL of supernatant.
    3.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    3.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    3.8
    Carefully remove and discard the ethanol.
    3.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    3.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    3.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    3.12
    Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated. 
    Caution: over-drying the beads may result in reduced yield.

  4. DNase Digestion
    To remove the hybridization oligo-nucleotides, the ribosomal depleted sample is incubated with DNase. Ensure that the DNase digestion master mix (Table 3) is prepared and kept at room temperature.
    4.1
    Assemble DNase digestion reactions as follows:


    4.2
    Thoroughly resuspend the beads by pipetting up and down multiple times.
    4.3
    Incubate the plate/tube(s) at room temperature for 3 min to elute the RNA off the beads.
    4.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    4.5
    Carefully transfer 20 μL of supernatant into a new plate/tube(s). Discard the plate/tube(s) with beads.
    4.6
    Incubate the plate/tube(s) with supernatant using the following protocol:


    4.7
    Proceed immediately to DNase Digestion Cleanup (step 5).

  5. DNase Digestion Cleanup
    5.1
    Perform a 2.2X bead-based cleanup by combining the following:


    5.2
    Thoroughly resuspend the beads by pipetting up and down multiple times.
    5.3
    Incubate the plate/tube(s) at room temperature for 5 min to bind the RNA to the beads.
    5.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    5.5
    Carefully remove and discard 60 μL of supernatant.
    5.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    5.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    5.8
    Carefully remove and discard the ethanol.
    5.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    5.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    5.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    5.12
    Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated.    
    Caution: over-drying the beads may result in reduced yield.
    5.13
    Thoroughly resuspend the beads (with purified, DNase-treated RNA) in an appropriate volume of RNase-free water by pipetting up and down multiple times.
    5.14
    Incubate the plate/tube(s) at room temperature for 3 min to elute RNA off the beads.
    5.15
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    5.16
    Carefully transfer the appropriate volume of supernatant into a new plate/tube(s). Discard the plate/tube(s) with beads. Store rRNA-depleted samples at -80°C.
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