Extracting RNA from LCM-d Porites

Full Extraction protocol: here

Short-hand protocol for reference

  • changes to standard: 65 uL digestion buffer instead of 60 uL to account for cel collection in 35 uL instead of 40 uL
  • digestion changed from 15 minutes room temp to 15 mins on thermomixer at 56 ºC shaking at 1400 rpm
  1. Thaw LCM-dissection tubes from -80 ºC for 5-10s at room temp.
  2. Once thawed, immediately add 65 uL of the digestion buffer and pipette up and down to mix with wide bore tip
  3. Incubate for 15 mins on thermomixer at 56 ºC shaking at 1400 rpm
    1. Nest the PCR tube into a 1.5mL tube
  4. Thaw an aliquot of DNase I from -20 ºC on ice
  5. Transfer entire volume to a 1.5 mL tube with wide-bore tip
  6. Spin at 9,000 rcf for 3.5 mins to pellet any debris, then move 95 uL of supernatant to new 1.5 mL tube.
  7. Add 190 uL RNA lysis buffer and mix well until clear
  8. Add an equal volume of 100% ethanol (285 uL) and mix well
    1. do not spin down or allow a precipitate to form
  9. Transfer entire volume into RNA (IC) column
  10. Spin column at 15,000 rcf for 30s, discard flow-through
    1. All spins, unless noted, were performed at 15,000 rcf for 30s
  11. Wash column with 400 uL wash buffer and spin down, discard flow through
  12. Prepare DNase treatment (5 uL DNase I with 35 uL DNA Digestion Buffer per sample) on ice
    1. perform DNAse treatment (40 uL) as written:
      1. apply 40 uL of the mixture directly to each filter
      2. incubate at room temperature for 15 minutes
  13. Add 400 uL prep buffer and spin down, discard flow through
  14. Add 700 uL wash buffer and spin down, discard flow through
  15. Add 400 uL wash buffer and spin for 2 minutes at 15,000 rcf
  16. Transfer column to labelled 1.5 mL tube and elute in 15 uL RNase/DNase-free water.
  17. Allow filter to saturate by spinning at 100 rcf for 1 minute and then elute by spinning down at 12,000 rcf for 1 minute.
  18. Confirm all liquid has been eluted from the filter into the tube, and discard the filter column.
  19. Immediately transfer RNA tubes to ice and perform QC (hsRNA tapestation using 2 uL RNA) as soon as possible.
    1. I recommend doing the RNA tapestation before continuing with the DNA extraction
  20. Store RNA at -80 ºC as quickly after the extraction as possible and limit freeze-thaw cycles.

Quality Control (QC)

High Sensitvity RNA Tapestation. Follow the Tape Station Protocol.

Quick reference:

  1. For each sample, pipette 1 µL High Sensitivity RNA Sample Buffer and 2 µL RNA sample in a tube strip
  2. Apply caps to tube strips.
  3. Mix liquids using the IKA MS3 vortexer at 2000 rpm for 1 min.
  4. Spin down samples and ladder for 1 min.
  5. Samples and ladder denaturation:
    1. Heat samples and ladder at 72 °C (162 °F) for 3 min.
    2. Place samples and ladder on ice for 2 min.
    3. Spin down sample and ladder for 1 min.
Written on June 21, 2026