RNA extraction

CTAB RNA extraction

  • For each 0.5 g sample, add 20 ul b-mercaptoethanol (b-ME) to 10 ml CTAB and pre-warm in a 65 °C water bath
  • b-ME is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds and destroying the native conformation required for enzyme functionality
  • Grind tissue in a pre-chilled mortar and pestle (oven-baked at 180 °C for > 2 hours) to fine powder under liquid nitrogen. Transfer ground sample to a pre-chilled RNase-free 50 ml Falcon tube. Add pre-warmed CTAB buffer (w/ b-ME), vortex for 1 min, and incubate for 30 min at 65 °C in a water bath. Vortex or shake vigorously by hand every 5 min.
  • Merely autoclaving will not destroy all RNase activity, since these enzymes are very robust and can regain partial activity upon cooling to RT
  • Always use tips, tubes, and H2O that have been tested and certified RNase-free
  • Add 10 ml of chloroform/IAA (24:1) and vortex for 1 min. Centrifuge at RT for 15 min at 5,000 rpm (20 min at 3,000 rpm works well)
  • Transfer the aqueous supernatant to another 10 ml of chloroform/IAA in a 50 ml Falcon. Vortex and centrifuge once more as above
  • Transfer supernatant to new Falcon tube being careful not to touch the white layer (usually 8 ml), and add 0.5 volume 96-100% ethanol. Gently mix by pipetting and transfer 750 ul of mixture to a RNeasy spin (PINK) column (Qiagen RNeasy Mini extraction kit). Centrifuge at max speed (13,500 rpm) for 30 seconds at RT. Discard flow-through.
    • Pass multiple volumes of supernatant-ethanol mixture through single RNeasy column to concentrate extract
  • Add 700 ul Buffer RW1 to the RNeasy column. Centrifuge for 30 sec at 13,500 rpm. Discard flow-through and collection tube
  • Transfer the RNeasy column into a new 2 ml collection tube. Add 500 ul Buffer RPE on to the RNeasy column. Centrifuge for 30 sec at 13,500 rpm. Discard flow-through
  • Repeat with 500 ul RPE, but centrifuge for 2 min at 13,500 rpm
  • Transfer the RNeasy column to a RNase-free 1.5 ml tube. Add 50 ul RNase-free water directly onto the RNeasy column membrane. Centrifuge for 2 min at 13,500 rpm. Repeat with another 50 ul RNase-free water or with elute from previous step if increased RNA concentration is desired.

 

DNase treatment (w/ TURBO DNA-free kit)

  • Start with 50 ul RNA sample
  • Add 0.1 volume 10X TURBO DNase Buffer and 1 ul TURBO DNase to the RNA, and mix gently
  • Incubate at 37 °C for 20-30 min
  • Add resuspended DNase Inactivation Reagent (typically 0.1 volume) at mix well
  • Incubate 5 min at RT, mixing occationally
  • Centrifuge at 10,000 x g for 1.5 min and transfer the RNA to a fresh tube
  • Usually ~50 ul
  • Perform NanoDrop analysis to estimate its concentration
  • A maximum of 100 ug RNA can be cleaned up in the following step
  • The RNA concentration is usually around 1,000 ng/ul after this step

 

RNA cleanup (w/ Qiagen RNeasy Mini extraction kit)

  • Adjust the sample to a volume of 100 ul with RNase-free water. Add 350 ul Buffer RLT, and mix well
  • Add 250 ul ethanol (96-100%) to the diluted RNA, and mix well by pipetting. Do not centrifuge. Proceed immediately to the next step
  • Transfer the sample (700 ul) to an RNeasy Mini spin column placed in a 2 ml collection tube. Close the lid gently, and centrifuge for 15 s at >= 8,000 x g (>= 10,000 rpm). Discard the flow-through
  • After centrifuge, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely
  • Add 500 ul Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s >= 10,000 rpm to wash the spin column membrane. Discard the flow-through
  • Add 500 ul Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min >= 10,000 rpm to wash the spin column membrane
    • The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elusion
  • Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min
  • Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30-50 ul RNase-free directly to the spin column membrane. Close the lid gently, and centrifuge for 1min at >= 10,000 rpm to elute the RNA.
  • If the expectedly RNA yield is > 30 ug, repeat the previous step using another 30-50 ul RNase-free water, or using the eluate from the previous step
  • Usually I used 40 ul water twice to elute the RNA (80 ul in total)
  • Perform NanoDrop analysis, electrophoresis, and Qubit analysis for each sample
  • NanoDrop will let you know the if there is any contamination of each sample
  • The concentration is usually around 500 ng/ul after clean up
  • Qubit HS RNA concentration is more accurate
    • The range of sample concentration for Qubit is 250 pg/ul to 100 ng/ul
    • Therefore, dilute the original sample 1:10 before performing Qubit analysis
  • Use 1 ul RNA sample for electrophoresis; 120 V, 1% gel, 35 min. If there are clearly two bands, 18S and 25S rRNA, this means the RNA is not degraded.