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  • High-throughput RNA isolation from FFPE samples
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NucleoSpin 96 DNA FFPE product page NucleoSpin 96 DNA FFPE
User-generated protocol

High-throughput RNA isolation from FFPE samples using the NucleoMag DNA FFPE kit

Introduction Materials required Protocol

Introduction  

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.

Materials required  

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.

NOTE: Each of these working solutions can be stored at −20°C for at least six months. Do not freeze/thaw the rDNase working solution more than three times.

Protocol  

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.

NOTES:

  • Make sure that the paraffin completely melts during the heat incubation step and mix well after melting to fully dissolve the paraffin.
  • Insufficient mixing of the heated sample may cause solid paraffin particles to reappear. Make sure the sample does not contain more than 15 mg paraffin or adjust the volume of Paraffin Dissolver.

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.

NOTE: The NucleoMag DNA FFPE kit protocol for RNA isolation does not include a decrosslinking step, unlike the DNA isolation protocol.

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

NOTE: Yield can be increased by 15–20% by using prewarmed elution buffer (55°C) or by incubating the bead/elution buffer suspension for 10 min at 55°C.

  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.

Related Products

Cat. # Product Size Price License Quantity Details
744320.1 NucleoMag® DNA FFPE 1 x 96 Preps USD $520.00

NucleoMag DNA FFPE kits employ superparamagnetic beads to enable high-throughput DNA purification from formalin-fixed, paraffin-embedded (FFPE) samples using manual or automated processing. The kits can be used to process fresh or archived samples and are compatible with inputs of ≤5 mg of tissue and ≤15 mg of paraffin. Purified DNA fragments typically range in size from 50 bp–5 kbp and are eluted in volumes >25 µl. The kits include blue-colored Paraffin Dissolver for convenient paraffin removal and enable processing of 96 samples in ~2 hours.

Cat. # 744320.1 includes sufficient reagents and materials for processing of 96 samples.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents You May Also Like Image Data

Back

744320.1: NucleoMag DNA FFPE

744320.1: NucleoMag DNA FFPE

Back

Competitor A (A) and competitor I (B) show higher CT values, indicating a lower yield compared to samples purified with NucleoMag DNA FFPE.

Competitor A (A) and competitor I (B) show higher CT values, indicating a lower yield compared to samples purified with NucleoMag DNA FFPE.

NucleoMag DNA FFPE provides better PCR performance than competitor kits. Genomic DNA was purified from FFPE samples using NucleoMag DNA FFPE in comparison with kits from Competitor A and Competitor I and analyzed using a LightCycler PCR system and the DyNAmo Capillary SYBR Green qPCR Kit (amplicon size: 191 bp). qPCR of DNA purified with kits from Competitor A (Panel A) and Competitor I (Panel B) resulted in higher Ct values, indicating lower DNA yields compared to samples purified with NucleoMag DNA FFPE.

Back

The NucleoMag DNA FFPE kit leverages the reversible adsorption of nucleic acids on paramagnetic beads to allow a quick, simple workflow for high-purity genomic DNA from FFPE tissue.

The NucleoMag DNA FFPE kit leverages the reversible adsorption of nucleic acids on paramagnetic beads to allow a quick, simple workflow for high-purity genomic DNA from FFPE tissue.

The NucleoMag DNA FFPE procedure.

Back

The NucleoMag DNA FFPE kit provides higher DNA yields from embedded mouse lung, liver, and kidney sections compared to competitor kits (A, I).

The NucleoMag DNA FFPE kit provides higher DNA yields from embedded mouse lung, liver, and kidney sections compared to competitor kits (A, I).

NucleoMag DNA FFPE provides higher yields than competitor kits. Genomic DNA was purified in triplicate from embedded mouse lung, liver, and kidney tissue sections using NucleoMag DNA FFPE and two competitor kits. The NucleoMag DNA FFPE kit provided higher DNA yields than the competitor kits (A, I).

744320.4 NucleoMag® DNA FFPE 4 x 96 Preps USD $1810.00

NucleoMag DNA FFPE kits employ superparamagnetic beads to enable high-throughput DNA purification from formalin-fixed, paraffin-embedded (FFPE) samples using manual or automated processing. The kits can be used to process fresh or archived samples and are compatible with inputs of ≤5 mg of tissue and ≤15 mg of paraffin. Purified DNA fragments typically range in size from 50 bp–5 kbp and are eluted in volumes >25 µl. The kits include blue-colored Paraffin Dissolver for convenient paraffin removal and enable processing of 96 samples in ~2 hours.

Cat. # 744320.4 includes sufficient reagents and materials for processing of 4 x 96 samples.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents You May Also Like Image Data

Back

Competitor A (A) and competitor I (B) show higher CT values, indicating a lower yield compared to samples purified with NucleoMag DNA FFPE.

Competitor A (A) and competitor I (B) show higher CT values, indicating a lower yield compared to samples purified with NucleoMag DNA FFPE.

NucleoMag DNA FFPE provides better PCR performance than competitor kits. Genomic DNA was purified from FFPE samples using NucleoMag DNA FFPE in comparison with kits from Competitor A and Competitor I and analyzed using a LightCycler PCR system and the DyNAmo Capillary SYBR Green qPCR Kit (amplicon size: 191 bp). qPCR of DNA purified with kits from Competitor A (Panel A) and Competitor I (Panel B) resulted in higher Ct values, indicating lower DNA yields compared to samples purified with NucleoMag DNA FFPE.

Back

The NucleoMag DNA FFPE kit leverages the reversible adsorption of nucleic acids on paramagnetic beads to allow a quick, simple workflow for high-purity genomic DNA from FFPE tissue.

The NucleoMag DNA FFPE kit leverages the reversible adsorption of nucleic acids on paramagnetic beads to allow a quick, simple workflow for high-purity genomic DNA from FFPE tissue.

The NucleoMag DNA FFPE procedure.

Back

The NucleoMag DNA FFPE kit provides higher DNA yields from embedded mouse lung, liver, and kidney sections compared to competitor kits (A, I).

The NucleoMag DNA FFPE kit provides higher DNA yields from embedded mouse lung, liver, and kidney sections compared to competitor kits (A, I).

NucleoMag DNA FFPE provides higher yields than competitor kits. Genomic DNA was purified in triplicate from embedded mouse lung, liver, and kidney tissue sections using NucleoMag DNA FFPE and two competitor kits. The NucleoMag DNA FFPE kit provided higher DNA yields than the competitor kits (A, I).

Back

744320.4: NucleoMag DNA FFPE

744320.4: NucleoMag DNA FFPE
740963 rDNase Set Each USD $97.00

The rDNase Set is intended for performing RNA minipreps using NucleoSpin RNA kits. Cat. # 740963 consists of one vial of RNase-free recombinant DNase (lyophilized), plus 7 ml Reaction Buffer.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents
744851.80 Buffer MB2 100 mL USD $65.00

Buffer MB2 is a binding buffer for isolation of genomic DNA that is included with various NucleoMag kits. Cat. # 744851.80 consists of a bottle containing 100 ml of buffer.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Image Data

Back

744851.80: Buffer MB2

744851.80: Buffer MB2
740834.60 Reaction Buffer for rDNase 60 mL USD $36.00

Reaction Buffer for rDNase is provided with many RNA purification kits as a reaction buffer for DNase-mediated DNA digestion. Cat. # 740834.60 consists of 60 ml of buffer.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Image Data

Back

740834.60: Reaction Buffer for rDNase

740834.60: Reaction Buffer for rDNase


User-generated protocols

User-generated protocols

User-generated protocols are based on internal proof-of-concept experiments, customer collaborations, and published literature. In some cases, relevant results are discussed in our research news BioView blog articles. While we expect these protocols to be successful in your hands, they may not be fully reviewed or optimized. We encourage you to contact us or refer to the published literature for more information about these user-generated and -reported protocols. 

If you are looking for a product-specific, fully optimized User Manual or Protocol-At-A-Glance, please visit the product's product page, open the item's product details row in the price table, and click Documents. More detailed instructions for locating documents are available on our website FAQs page.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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