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RNA Extraction from Red Algae

(Contributed by S. Ghoshroy, edited 11/15/2011 by DLR)

Preparation of material:

Read the following set of instructions before you plan to extract RNA.  Solutions, and glassware should be ready before you take the samples out of the freezer for extraction.  Before preparing solutions or starting extractions, metal scoops and spatulas, a mortar and pestle, a few beakers, Erlenmeyer flasks, and bottles should be baked at 275° C for 2 to 3 h.  Treating solutions and water with 0.1% diethyl pyrocarbonate (DEPC) can reduce RNAse contamination.  Compounds containing amine groups (e.g. Tris) cannot be DEPC and must be made using ultrapure stocks and DEPC water. Plasticware (blue (100-1000μl) and white (20-200μl) pipette tips, preloaded boxes, test tubes, etc.) untouched by humans should be autoclaved.

DEPC-treated water can be generated by treatment with 0.1% DEPC for 1 hour at 37ºC and autoclaving for 15-20 at 15 psi. For more information about working with RNA, we recommend reading “How to win the battle with RNase” in Sambrook and Russell, Molecular Cloning, Volume 1. 782-7.85.

 

Preparation of Solutions.

  1. Prepare 70% EtOH for RNA use with DEPC-treated water in RNase-free bottle or centrifuge tube.
  2. Preparation of solution D (denaturing solution) [Sambrook and Russel: Molecular Cloning. Volume 1, 7.4-7.8]:
    • 4M Guanidinium thiocyanate
    • 25mM Sodium citrate. 2H2O
    • 0.5% (w/v) Sodium lauryl sarcosinate
    • 0.1M β-mercaptoethanol.
  3.  For preparing 100 ml.
    • Add 47.28g of Guanidinium thiocyanate in 50 mL H2O
    • (Solution D is very caustic.   Wear appropriate gloves, a laboratory coat, and eye protection when preparing, handing, or working with this solution)
    • 5 ml of 10% sarcosyl
    • 3.3 ml of 0.75M sodium citrate pH 7
    • Bring the volume to 100 ml with DEPC-treated H2O
    • [Add β-mercaptoethanol to an aliquot of solution D before each use]

 

RNA extraction protocol

  1. Add liquid nitrogen to empty mortar and pestle, let it evaporate.
  2. Transfer all of the frozen tissue to a mortar containing liquid nitrogen and pulverize the tissue using a pestle.  The tissue can be kept frozen during pulverization by addition of liquid nitrogen.
  3. Transfer the pulverized tissue to a polypropylene snap cap tube (falcon tubes), containing 3 ml of solution D. Add 22μl of β-ME for 3ml of solution D.
    • 100 mg of tissue: 3 mL solution D. (Sambrook)
    • 100 mg of cells: 1mL  solution D (Lab protocol).
  4. Sonicate 10 times for 1sec on high.  This will vary with your sonciator. We use a microtip and set the sonicator on full power.  Alternatively, material can be broken up by bead beating.
  5. Sequentially add:
    • 0.3 ml of 2M NaOAc pH 4. Mix.
      [stock =3 M NaOAc.  Use 0.2mL of 3M NaOAc pH 4- following the lab protocol]
    • 3 mL  of phenol. Mix. (This should be around pH 6.0 – we use OmniPur Phenol (6705) without the addition of the supplied Tris:EDTA buffer)
    • 0.6 ml of chloroform: isoamyl alcohol (IAA) (49:1). (Prepare before you start extraction.)
  6. Mix well for 10 sec or longer.  Solution should appear uniformly mixed.
  7. Chill on ice for 15 min.(Turn on centrifuge, change rotors and cool to 4ºC).
  8. Centrifuge at 10,000g for 20 min. at 4ºC. RNA is present in the aqueous phase.
  9. Transfer the upper aqueous phase to new tube and add equal volume of chloroform: IAA (49:1). Mix well and repeat centrifugation at 10,000g for 20 min at 4ºC. Repeat these steps until the interface is clear.
    • [Note: To minimize contamination by DNA trapped at the interface, avoid taking the lowest part of the interface.]
  10. Transfer the aqueous phase to a clean tube. Add 3 mL of isopropanol. Place at -20ºC for 1 hr. [Depending on how much RNA precipitates you can decrease the time to ½ hr.]
  11. Centrifuge at 10,000 x g for 20 min at 4 ºC (30 min in Sambrook)
  12. Decant isopropanol. Air dry pellet. (Clean and dry RNA should be translucent).
    • [Important: Pellets are easily lost. Decant the supernatant into a fresh tube. Do not discard it until the pellet has been checked.]
  13. Dissolve the pellet in 0.3 ml of solution D.
  14. Add equal volume of chloroform: IAA, mix thoroughly and centrifuge at 10,000g for 20 min. at 4ºC.
  15. Transfer the upper aqueous phase in a clean 1.5 ml tube and precipitate with 1 volume of isopropanol. Store at -20ºC for 1 hr and centrifuge at 10,000g for 20 min. at 4ºC.
  16. Decant the supernatant and wash with 70% ethanol. (~600μl) [This will get rid of the salts that came along during precipitation.]
  17. Centrifuge at 10,000g for 20 min. at 4ºC. Decant the supernatant and air dry the pellet.
  18. Dissolve the pellet it DEPC-H2O (use your own judgment depending on the pellet. ~50-100μl) and store at -80ºC.