4.3.2. Bulk extraction of RNA from 50-100 whole bees using the acid-phenol method

For colony-level surveys of bee microbes, including pathogens, it is often important to analyse a bulk sample of bees in order to ensure a more accurate view of colony loads (most parasites and pathogens are not found uniformly across all bees in the hive, see section 4. ‘Obtaining adult workers for laboratory experiments’ of the BEEBOOK paper on maintaining adult workers in vitro laboratory conditions (Williams et al., 2013) and the BEEBOOK paper on statistics (Pirk et al., 2013) for details on how to sample bees). Similarly, if a colony-level genetic or phenotypic (gene-expression) trait is desired it is often better to generate an estimate that is the average across many colony members rather than a few selected bees. Extractions from a sample of tens of bees can be costly since volumes of reagents must be scaled up. The below protocol greatly reduces the most costly (and hazardous) ingredient used in RNA extractions, acid-phenol, and otherwise generates equivalent yields and purity to the TRIzol® extraction described above.

  1. Put whole frozen bees (stored at -80oC since death) into a disposable extraction bag (e.g. www.Bioreba.ch) and add 500ml lysis/stabilization solution (section 4.3.3) per bee (i.e. for 50 bees add 25ml solution).
  2. Mash until homogenized using a rolling pin, leaving the bag partly open initially to allow air to escape.
  3. Allow to settle ~10 min so bubbles go down.
    You can mash 10 or so bags consecutively at a time. By the time #10 is finished, the bubbles in #1 have subsided. Keep pending bags on ice in bucket.
  4. Transfer 620ml of extraction liquid into a pre-labelled 1.5 ml micro tube.
    Note: It is advisable to save subsamples of the lysed tissues as a reserve (Store at-80°C).
  5. Add 380ml acid phenol.
  6. Vortex 30 sec to mix well.
  7. Incubate 10 min in a 95°C hot block.
    Place weight on top of tubes to prevent lids from popping open.
  8. Wearing goggles and a lab coat carefully remove weight and then transfer the tubes from hot block to pre-chilled rack in ice.
    It is best to keep hot block in hood to contain the phenol.
  9. Incubate on ice for 20 min.
  10. Bring to RT.
  11. Add 200ml chloroform. 
  12. Shake vigorously for 1 sec.
  13. Incubate at RT 3 min.
  14. Centrifuge at 14,000 rpm for 15 min at 4°C.
  15. Transfer 500ml upper phase to fresh tube.
  16. Add equal volume of isopropanol (100%).
  17. Invert ten times to mix.
  18. Incubate at RT 15 min.
  19. Centrifuge at 10,000 rpm for 10 min at 4°C.
  20. Carefully decant liquid from pellet.
  21. Wash w/ 1ml of cold 75% EtOH.
  22. Centrifuge at 10,000 rpms for 2 min at 4°C.
  23. Carefully decant liquid from pellet.
  24. Spin 1 min.
  25. Remove excess alcohol with pipette tip.
  26. Air dry completely.
  27. Resuspend in 200ml nuclease-free H2O.
  28. Solubilize for 10 min at 55°C.
  29. Store at -80°C.
  30. Yields should be higher than 200 mg (1 mg/ml) total RNA, and extractions should be stable for > 5 years. RNA degradation can be checked using an Agilent Bioanalyzer or by 2% agarose gels looking for the co-migrating large ribosomal RNA’s as a sign of largely intact RNA. If extractions are to be shipped or kept at temperatures above -50oC for more than 48 hours, RNA should first be precipitated in an equal volume of isopropyl alcohol, shipped in that state, then suspended starting at step 22 above.