NucleoMag DNA FFPE, a magnetic bead-based kit designed for isolating DNA from formalin-fixed, paraffin-embedded (FFPE) samples in a 96-well format, may also be used to isolate RNA from FFPE samples using the following alternative protocol, which is adapted from the NucleoMag DNA FFPE Genomic DNA Purification User Manual.

Equipment/Consumables

• Magnetic separation system (e.g., NucleoMag SEP, Cat. # 744900)
• Separation plate for magnetic beads separation (e.g., Square-well Block; Cat. # 740481, 740481.24)
• Lysis tubes for incubation of samples and lysis (e.g., Rack of Tubes Strips, Cat. # 740477, 740477.24 or 1.5-ml microfuge tubes)
• Elution plate for collecting purified nucleic acids (e.g., Elution Plate U-bottom; Cat. # 740673, 740486)
• Accessories for use of kit on the KingFisher Flex instrument (e.g., KingFisher 96 Accessory Kit A; Cat. # 744950)

Reagents

• NucleoMag DNA FFPE kit (Cat. # 744320.1, 744320.4)
  
  
  • NucleoMag B-Beads
  • Lysis Buffer FL
  • Binding Buffer MB2 (available separately as Cat. # 744851.80)
  • Wash Buffer MB4
  • Elution Buffer MB6
  • Proteinase K (lyophilized)*
  • Proteinase Buffer PB
  • Paraffin Dissolver (blue)
  • Decrosslink Buffer D-Link
• rDNAse Set (Cat. # 740963)
  
  
  • rDNase, RNase-free (lyophilized)*
  • Reaction Buffer for rDNase (available separately as Cat. # 740834.60)

• 80% ethanol

*Prior to beginning the protocol, prepare working solutions of Proteinase K and rDNase as described below:

Preparation of Proteinase K solution

Before first use, add 2.8 ml of Proteinase Buffer PB to a vial (75 mg) of lyophilized Proteinase K to dissolve the Proteinase K.

Preparation of rDNase reaction mixture

1. Reconstitution of lyophilized rDNase: Before first use, add 4 ml of Reaction Buffer for rDNase to the rDNase vial and incubate for 2–3 min at room temperature. Gently swirl the vial to completely dissolve the rDNase. Be careful not to mix rDNase vigorously as rDNase is sensitive to mechanical agitation.
2. Dilution of rDNase: Transfer the reconstituted rDNase to a suitable tube and add 28 ml of Reaction Buffer for rDNase. Gently swirl the tube. The resulting rDNase reaction will be sufficient for 96 samples. Prepare a smaller amount (e.g., 1 ml reconstituted of rDNase and 7 ml of Reaction Buffer for rDNase for 32 reactions), when performing fewer reactions. For each isolation, combine 37.5 μl of reconstituted rDNase and 262.5 μl of Reaction Buffer for rDNase.

This protocol is designed for use with a magnetic separator with static pins (e.g., NucleoMag SEP) and a plate shaker whose speed can be adjusted to prevent cross-contamination from well to well. We recommend using a Square-well Block for separation. Alternatively, isolation of DNA can be performed in 1.5-ml microfuge tubes with suitable magnetic separators. This protocol is for manual use and serves as a guideline for adapting the kit to robotic instruments.

Deparaffinize samples

1. Add each sample to a 1.5-microfuge tube.
2. Add 400 µl of Paraffin Dissolver to each sample and incubate them for 3 min at 60°C (to melt the paraffin).
3. Vortex or shake the samples immediately (at 60°C) at a vigorous speed to dissolve the paraffin, and then allow them to cool to room temperature.

Lyse samples and transfer to Square-well Block

1. Add 200 μl of Lysis Buffer FL and 25 μl of Proteinase K solution to the lower aqueous phase of the samples and mix well by repeated pipetting up and down, pulse vortexing, or shaking.
2. Centrifuge the samples for 1 min at 11,000g and incubate them for a maximum of 90 min at 56°C.
3. Centrifuge the samples again for 1 min at 11,000g.
4. Transfer up to 400 μl of the lower aqueous phase from each sample to a Square-well Block well for further processing.

Bind DNA to NucleoMag B-Beads

1. Resuspend the NucleoMag B-Beads before removing them from the storage bottle. Vortex the storage bottle briefly until a homogenous suspension has been formed.
2. Add 14 μl of NucleoMag B-Beads and 600 μl of Binding Buffer MB2 to each of the lysed samples.
3. Mix by pipetting up and down ten times and incubate for 5 min at room temperature.
4. Separate the magnetic beads against the sides of the wells by placing the Square-well Block on the NucleoMag SEP magnetic separator. Wait at least 2 min until all the beads have been attracted to the magnets. Then remove and discard the supernatant by pipetting.
   
   Important: Do not disturb the attracted beads while removing the supernatant.
5. Dry beads for 5 min at room temperature.

Digest DNA

1. Remove the Square-well Block from the NucleoMag SEP magnetic separator.
2. Add 300 μl of rDNase reaction mixture to each bead pellet and resuspend the beads by pipetting up and down.
3. Incubate for 15 min at room temperature. Do not separate the beads.

Rebind

1. Add 350 μl of Binding Buffer MB2 to each sample. Mix by shaking for 5 min at room temperature or by pipetting up and down six times. Perform a subsequent incubation for 5 min at room temperature.
2. Separate the magnetic beads against the side of the wells by placing the Square-well Block on the NucleoMag SEP magnetic separator. Wait at least 2 min until all the beads have been attracted to the magnets. Remove and discard the supernatant by pipetting.

Wash with MB4

1. Remove the Square-well Block from the NucleoMag SEP magnetic separator.
2. Add 600 μl of Wash Buffer MB4 to each well and completely resuspend the beads by shaking (1–3 min) or by repeated pipetting up and down (15 times).
3. Separate the magnetic beads by placing the Square-well Block on the NucleoMag SEP magnetic separator. Wait at least 2 min until all the beads have been attracted to the magnet. Remove and discard supernatant by pipetting.

First wash with 80% ethanol

1. Remove the Square-well Block from the NucleoMag SEP magnetic separator.
2. Add 600 μl of 80% ethanol to each well and completely resuspend the beads by shaking (1–3 min) or by repeated pipetting up and down (15 times).
3. Separate the magnetic beads by placing the Square-well Block on the NucleoMag SEP magnetic separator. Wait at least 2 min until all the beads have been attracted to the magnet. Remove and discard supernatant by pipetting.

Second wash with 80% ethanol

1. Remove the Square-well Block from the NucleoMag SEP magnetic separator.
2. Add 600 μl of 80% ethanol to each well and completely resuspend the beads by shaking (1–3 min) or by repeated pipetting up and down (15 times).
3. Separate the magnetic beads by placing the Square-well Block on the NucleoMag SEP magnetic separator. Wait at least 2 min until all the beads have been attracted to the magnet. Remove and discard supernatant by pipetting.
4. Air dry the magnetic bead pellet for 10 min at room temperature.

Elute

1. Add the desired volume of Elution Buffer MB6 (25–100 μl) to each well of the Square-well Block and resuspend the beads by shaking 5 min at room temperature or by repeated pipetting up and down. Incubate the bead suspensions for 10 min at 56°C.
2. Separate the magnetic beads by placing the Square-well Block on the NucleoMag SEP magnetic separator. Wait at least 2 min until all the beads have been attracted to the magnets. Then transfer the supernatants containing the purified genomic DNA to either 1.5-ml microfuge tubes or Tube Strips.