RNA, DNA and Protein Content Protocol

GLOBEC Georges Bank Program, Broadscale Survey Protocol for sampling larval cod (Gadus morhua) and haddock (Melannogrammus aeglefinus) at sea to be analyzed for RNA, DNA, and protein content.
NOTE: The overriding consideration in all of the following steps is that the fish should be kept as cold as possible during all the steps, and the fish should be processed as quickly as possible at all times. The fish are shrinking and degrading from the time they are first captured in the net until they are frozen in liquid nitrogen, so the shorter the time between capture and freezing the better the quality of the samples.
  1. One oblique bongo net tow per station.
  2. Maintain boat speed at 1.0 - 2.0 knots (average 1.5 knots) trying to maintain 45 degree wire angle.
  3. Tow the bongo a minimum of 5 minutes to a maximum of 18-20 minutes, depending on the depth of the water at the station. See attached sheet for wire times.
  4. Bongo is equipped with .333 mm mesh nets.
  5. When nets come on board rinse entire net gently but thoroughly with seawater using the deck hose.
  6. Remove cod end bucket and pour contents into white bucket labeled Buckley.
  7. Immediately bring the sample bucket into the dry-lab for sieving and sorting.
  8. Pour contents of bucket through a .333 mm mesh sieve using seawater to wash contents (or a .505 mm mesh sieve if that mesh net was used for that tow).
  9. Sort through sieved contents using lighted magnifying glass or sorting trays and light table.
  10. Place cod and haddock in separate prelabeled petri dishes filled with seawater and placed on freezer packs.
  11. Place fish of one species only on a microscope slide
  12. Record station location, tow number, mesh size, magnification, species, and dewar slot/shelf on data sheet.
  13. Assign next consecutive number from data sheet to each fish.
  14. Take video of fish, record fish number by voice.
  15. Immediately place each fish in an eppendorf tube previously labeled with the consecutive numbers and store in blue ice rack. When all fish from one tow are videotaped or the rack is full, place eppendorfs in liquid nitrogen dewar.
  16. Keep blue ice racks and freezer packs separated by watch so that they will have sufficient time to freeze before they are used again.
  17. Check level of liquid nitrogen in dewars daily. As needed, add liquid nitrogen from the 200 liter stock tank to maintain a 1-2 inch liquid level in the bottom of the dewar.
Revised: 01/09/95 E. Caldarone