Charm Biotech Just-a-Tube ™ Laser Captured Microdissection (LCM) Sample Total RNA/MicroRNA Purification Kit

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

Materials and Equipment

  • Charm Biotech Just-a-Tube ™ Laser Captured Microdissection (LCM) Sample Total RNA/MicroRNA Purification Kit, protocol
  • Heating block capable of heating to 60ºC
  • Centrifuge and rotor capable of spinning at 15,000 rcf
  • 14.3 M B-mercaptoethanol
  • 100% Ethanol
  • 100% Isopropanol
  • DNAse I and its buffer
  • 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 the sample kit, add 5 mL of ethanol to WB2.

Sterilizing working area to maintain an RNAse-free 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
  4. RNAse cleaner (spray bottle)

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. Repeat with RNAse cleaner. Wipe down gloves with 70% ethanol and RNAse cleaner. 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 (RD1) if you use LCM samples from frozen tissue sections, or fresh working Digestion Buffer RD2 (RD2)if you use LCM samples from FFPE tissue sections.
    • Add 9 ul of Proteinase K (provided) and 1 ul of 14.3 M B-mercaptoethanol (BME, not provided) or 1.0M DTT (not provided) to each 90 ul of Digestion Buffer (RD1 or RD2) to reach final 1% (v/v)B-mercaptoethanol or 10 mM DTT concentration and mix well. Add 50 ul working Digestion Buffer for each LCM sample.
      • For 2 samples: 90 ul RD2 + 9 ul Proteinase K + 1 ul BME
        • split into 2 tubes, 50 uL each
      • For 4 samples: 190 ul RD2 + 18 ul Proteinase K + 2 ul BME
        • split into 4 tubes, 50 uL each
  • Prepare fresh working Binding Buffer (RB7) prior to performing RNA isolation procedure based on the number of samples processed. To make fresh working Binding Buffer (RB7), for each 10 ul of Binding Buffer (RB7) add 40 ul of 100% isopropanol. Mix well
    • Prepare a master working Binding Buffer solution based on (1) the number of samples processed and (2) any anticipated loss, generally 10%, during dispensing. Dispense 100 ul of Binding Bufer containing isopropanol per 50-ul final cell lysates. Discard the unused Binding Buffer at the end of the day.
      • For 2 samples: 176 ul 100% isopropanol + 44 ul RB7
        • makes 210 ul, enough for 2 samples + 10% error
      • For 4 samples: 352 ul 100% isopropanol + 88 ul RB7
        • makes 440 ul, enough for 4 samples + 10% error

Protocol Steps

  1. Sample prep from LCM samples
    • Add 50 uL of prepared digestion buffer (see above) 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.
      • Frozen tissue protocol: use RD1, prepared as detailed above
      • FFPE protocol: use RD2, prepared as detailed above
  2. Incubate the inverted tube at 52 ºC for frozen tissues or at 6 ºC for FFPE tissues for 10-15 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. Incubate the tube at 52 ºC or 60 ºC in a thermocycler or incubator for complete cell/tissue digestion.
    • Frozen tissue protocol: Incubate at 52 °C for 1-2 hour with occasional mixing until lysis is complete.
    • FFPE protocol: Incubate at 60 ºC for 3-4 hour with occasional mixing until lysis is complete.
      • (Optional: For FFPE tissue samples, you may perform digestion at 48 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 briefly to collect all liquid at the bottom of the tube.

Binding RNA Products

  1. Transfer all 50 ul cell lysate solution from the capturing tube to a Binding Tube (BT5).
  2. Add 100 uL fresh RB7 (prepared above) to the Binding Tube (BT5). Mix well with the lysate by pipeting up and down the solution 10 times to obtain a homogenous solution.
  3. Centrifuge the Binding Tube (BT5) at maximum speed (about 13000 rpm or 16000 X g) at room temperature for 8 minutes ot bind the RNA
    • (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 200 ul Wash Buffer II (WB2) containing ethanol to the Binding Tube (BT5). Incubate the tube for 30 seconds at room temperature.
  2. Centrifuge the Binding Tube (BT5) 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 (BT5) 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 (BT5) 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.

DNase I Digestion, and Eluting RNA Products

Sample Elution

  • RNA attached to the walls of the Binding Tube can be stored directly in the tube at -80 °C for long-term storage until further use. If the RNA is to be eluted for analysis before DNase I digestion, please follow the procedure below.
  1. Add 20 ul Elution Buffer (EB3) into each tube.
  2. Close the tube and vortex the tube for 10 - 30 seconds, or tap the tube 10 - 15 times with a finger.
  3. Briefly centrifuge the tube to collect all solution, and place the tube on ice for downstream applications. If you are not going to analyze the samples immediately, store the tube at -80 °C until use.

If DNaseI digestion procedure is needed, please directly proceed to the DNase I digestion procedure without sample elution first.

DNase I Digestion

To prevent any DNA contamination, DNase I (not provided) digestion is required for removal of gDNA. When choosing DNase I, please make sure to use RNase-free DNase I.

  1. DNase I digestions can be performed directly in the Binding Tube. For DNase I digestion, use a final reaction volume of 20 ul for each sample.
    • Please follow the DNase I supplier’s instructions for DNase I digestion and inactivation directly in the Binding Tube. (Note: After inactivation of DNase, RNA sample can be used directly for downstream applications.)
  2. Once the DNase I digestion and inactivation are completed, centrifuge the Binding Tube briefly and place it on ice for further downstream applications. If you are not going ot analyze the samples immediately, store the tube at -80 ºC until use

Quality Control (QC)

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

RNA Quantity

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

RNA Quality

Proceed to this step if RNA does not appear clean on the gel. If RNA quantity is sufficient follow the Tape Station Protocol to determine RNA quality and obtain a RNA Integrity Number (RIN). “Good” RNA should have a RIN above 8.0 and form two distinct peaks at the 18S and 28S locations.

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 25, 2024