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.
- One oblique bongo net tow per station.
- Maintain boat speed at 1.0 - 2.0 knots (average 1.5
knots) trying to maintain 45 degree wire angle.
- 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.
Bongo is equipped with .333 mm mesh nets.
- At the deep stations, deploy the nets (wire out) at a
maximum rate of 50 m/min and retrieve at a maximum
rate of 20 m/min. The net fishes top 200m of water
- In shallow water (200m or less) the net fishes the
entire water column and the retrieval rate should be
slowed to fish the net a minimum of 5 minutes. (5
minutes provides the volume of water previously
determined to be the minimum needed to be fished to
obtain statistically valid data).
When nets come on board rinse entire net gently but
thoroughly with seawater using the deck hose.
Remove cod end bucket and pour contents into white bucket
Immediately bring the sample bucket into the dry-lab for
sieving and sorting.
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).
- If .333 mm mesh clogs due to high abundance of
phytoplankton at a particular station or area then
switch to .505 mm mesh nets at those stations.
Sort through sieved contents using lighted magnifying
glass or sorting trays and light table.
Place cod and haddock in separate prelabeled petri
dishes filled with seawater and placed on freezer packs.
- If volume in bucket is large, sieve and sort through
one-half or one-third of contents of the bucket at a
time. Put two freezer packs in a zip-lock bag in the
bucket to keep the remainder of the sample cold.
Place fish of one species only on a microscope slide
Record station location, tow number, mesh size,
magnification, species, and dewar slot/shelf on data sheet.
Assign next consecutive number from data sheet to each
Take video of fish, record fish number by voice.
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.
Keep blue ice racks and freezer packs separated by watch
so that they will have sufficient time to freeze before they
are used again.
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
- Try to handle fish gently - forceps can cut and squish