Charm Biotech Just-a-Tube ™ Laser Captured Microdissection (LCM) Sample Genomic DNA Purification Kit

Goal: to extract high-quality gDNA from PAXgene-fixed cryosectioned tissue.

Materials and Equipment

  • Charm Biotech Just-a-Tube ™ Laser Captured Microdissection (LCM) Sample Genomic DNA Purification Kit, protocol
  • Heating block capable of heating to 60ºC
  • Centrifuge and rotor capable of spinning at 15,000 rcf
  • 100% Ethanol
  • Plastics: 0.2 mL PCR tube for LCM sample collection, 1-2 1.5 mL microcentrifuge tubes for mixing buffers, several 1000 uL, 200 uL, and 20 uL pipette tips per sample.

First time opening kit: Reagent Preparation

  • Add appropriate amount of ethanol for size of kit to Washing Buffer II (WB2).
    • For this kit, add 24 mL of ethanol to WB2.

Sterilizing working area to maintain a clean environment

Clean bench with clean paper towels (spray solution, wipe down) in the following order:

  1. 10% bleach solution
  2. DI water
  3. 70% ethanol

Clean pipettes, tip boxes, and the controls on the heating block and centrifuge by squirting 70% ethanol on a paper towel and wiping them down. Wipe down gloves with 70% ethanol. Do not spray solutions directly on the equipment.

Day of extraction (only prep amount for samples extracted that day): Reagent Preparation

  • Prepare fresh working Digestion Buffer RD1 (DD1) if you use LCM samples from frozen tissue sections (if using FFPE tissue, use buffer DD2 instead).
    • Add 10 ul of Proteinase K (provided) to each 90 ul DD1 for each LCM sample.
  • (Note: Digestion Buffer 1 (DDI) contains SDS, which can precipitate out of the buffer if stored at room temperature for a long time. If a precipitate is present, incubate the buffer in a 37 °C - 58 ºC water bath for 5 minutes, or until the SDS re-dissolves and the solution clears. Mix thoroughly but swirling gently.)

Protocol Steps

  1. Sample prep from LCM samples
    • LCM from frozen tissue sections
      • Add 100 uL of prepared buffer DD1 (see above) for LCM to the cap containing the LCM-captured cells, invert the tube and tap the cap with a finger until the solution spreads and covers the whole cap completely.
    • LCM from FFPE tissue sections
      • Add 100 uL buffer DD2
  2. Incubate the inverted tube at 60 ºC for 10 minutes to allow the captured cells and tissues detaching from the cap into the digestion buffer.
  3. Centrifuge briefly to collect all liquid at the bottom of the tube. Mix the liquid by pipetting up and down the solution several times to disperse any pellet precipitate.
  4. Digestion with Proteinase K
    • LCM from frozen tissue sections
    • Incubate at 60 °C for 3-4 hours with occasional mixing until lysis is complete.
      • (Optional: You may perform digestion at 52 ºC overnight if preferred).
    • LCM from FFPE tissue sections
      • Incubate at 94 ºC for 10 minutes, briefly spin tube to collect everything in the bottom
      • Add 10 uL proteinase K to the tube and mix well
      • Incubate at 60 °C for 3-4 hours with occasional mixing until lysis is complete.
        • (Optional: You may perform digestion at 52 ºC overnight if preferred).
    • At the end of incubation,no pellets should be visible, otherwise, extend incubation 15 minutes more to make sure no pellets exist.
  5. Centrifuge the microtube at maximum speed (about 13000 rpm or 16000 X g) for one minute.

Alternate version of this for Paxgene-fixed, cryoembedded samples (incubation does not need to be as aggressive because there is no paraffin and the tissue was fixed with Paxgene fixative, not formalin)

  1. Sample prep from LCM samples
    • LCM from PFCE tissue sections
      • Add 100 uL buffer DD2
  2. Incubate the inverted tube at 60 ºC for 10 minutes to allow the captured cells and tissues detaching from the cap into the digestion buffer.
  3. Centrifuge briefly to collect all liquid at the bottom of the tube. Mix the liquid by pipetting up and down the solution several times to disperse any pellet precipitate.
  4. Digestion with Proteinase K
    • LCM from PFCE tissue sections
      • Add 10 uL proteinase K to the tube and mix well
      • Incubate at 60 °C for 3-4 hours with occasional mixing until lysis is complete.
        • (Optional: You may perform digestion at 52 ºC overnight if preferred).
      • Alternatively, I have had success with a shorter incubation for RNA, see notes here
        • Instead of 60 °C for 3-4 hours, try shaker–incubator for 1 h at 56°C and 1400 rpm.
    • At the end of incubation,no pellets should be visible, otherwise, extend incubation 15 minutes more to make sure no pellets exist.
  5. Centrifuge the microtube at maximum speed (about 13000 rpm or 16000 X g) for one minute.

Binding DNA Products

  1. Transfer all 100 ul cell lysate solution from the capturing tube to a Binding Tube (BT3).
  2. Add 200 uL Binding Buffer (PB5) (prepared above) to the Binding Tube (BT3). Mix well with the lysate by pipeting up and down the solution 10 times to obtain a homogenous solution.
  3. Centrifuge the Binding Tube (BT3) at maximum speed (about 13000 rpm or 16000 X g) at room temperature for 5 minutes ot bind the DNA
    • (Note: During centrifugation always position the micro-tube hinge pointed outward from thecenter of rotation. Maiority of RNA will collect at the botom along the hinge side of the Binding Tube.)
  4. Remove the solution from the tube
    • Decant the solution by quick flip-over the tube over a waste container and shaking briskly, then put the inverted tube on a stack of clean absorbent paper such as Kimwipe, or paper towels, then tap the tube on the clean paper to remove as much liquid as possible
    • If pipetting the liquid out instead, be sure not to scrape the walls of the tube with pipette tips during aspiration as the products are bound to the walls of the Binding Tube.

Washing Products

  1. Add 250 ul Wash Buffer II (WB2) containing ethanol to the Binding Tube (BT3).
    • Mix by pipetting the solution up and down 2-3 times
    • Incubate the tube for 10-30 seconds at room temperature.
  2. Centrifuge the Binding Tube (BT3) at maximum speed (about 13000 rpm or 16000 X g) at room temperature for one minute. (Note: During centrifugation, always position micro-tube hinge pointed outward from the center of rotation.)
  3. Remove the WB2 from the Binding Tube as above.
  4. Repeat step 1 to 3 above for a total of two washes with Washing Buffer II (WB2).
  5. After the final wash, to ensure complete removal of Washing Buffer, spin the Binding Tube (BT3) very briefly, aspirate the last drop of liquid at the bottom of the tube with a 200 ul pipet tip, and air-dry the Binding Tube (BT3) in a lab hood for 8-10 minutes to remove any residual liquid.
    • Be sure not to scrape the walls of the tube with pipette tips during aspiration as the products are bound to the walls of the Binding Tube.

Eluting Products

Sample Elution

  1. Add 10-40 ul Elution Buffer (EB1) into each tube.
  2. Elution options
    1. Option #1 (Elute with vortex): Close the tube and vortex for 30 seconds. Centrifuge the tube at maximum speed briefly to collect all liguid at the bottom of each tube
    2. Option #2 (Elute with pipette): Pipette the solution up and down 5-7 times, and incubate for >60 seconds at room temperature
    3. Option #3 (Elute with shake): Close the tube and shake the tube for 5-6 minutes with a moderate-speed shaker.
  3. The eluted DNA may be used immediately in downstream applications. Alternatively, the eluted DNA may be stored at 4 °C for short-term storage or -20 °C for long-term storage.

Quality Control (QC)

These steps analyze the quantity and quality of the DNA extracted.

DNA Quantity

Follow Broad Range dsDNA Qubit protocol to analyze sample quantity. Read standards once and record values, read all samples twice.

DNA Quality

If DNA quantity is sufficient (typically >10 ng/µL) follow the PPP Agarose Gel Protocol to determine DNA quality. “Good” DNA should form a distinct band a the very top of the gel.

Sample prep notes for extracting from whole sections instead of LCM samples:

  1. Remove a cryosectioned slide from -80 ºC (same sample/sectioning round as used for LCM), transfer to -20 ºC for 30 minutes, then transfer slide to cooler with dry ice
    1. KEEP CONTAINER CLOSED, with dessicant inside
      • “take slide in container with desiccant out of -80 ºC and allow to slowly reach room temperature, for at least 60 min, before opening (Roy et al 2020 Bioprotocols)”
  2. Pre-cool lysis buffer mixture on ice
  3. Pre-cool forceps and razor blades for transferring tissue to lysis buffer
  4. Stain and wash all slides and perform dissections in petri dishes cleaned to be RNAse free
  5. Remove OCT, in RNAse free petri dish on ice, pipette solutions on and off slide with 1mL pipette
    • 2 minute in ice-cold 70% Ethanol
    • 45 s in ice-cold 50% Ethanol
    • 30 s in ice-cold 30% Ethanol
    • if there is excess OCT, dip slide in ice-cold RNAse free water 5-6 times over 1-2 mins
  6. Remove slide to dark background over PCR rack (over dry ice, though be careful to not freeze the water once the volume on the slide is low, if that’s freezing move to regular ice) and use sterile razor blade, cleaned with RNAse cleaner, to scrape off tissue sections into a tube filled with prepared 100 uL digestion buffer.
Written on January 31, 2024