Protocol for the preservation of animal tissues for RNA and DNA and Metabolomics samples in the presence or absence of liquid nitrogen and -80 freezers. This provides an option for remote field collection protocol.

Necessities

Reagents

• RNA/DNA Shield
• Ethanol 100%
• Bleach 10%
• DI water

Equipment and Consumables

• Stainless steel bone cutters/nail clippers
• Stainless steel forceps
• Ethanol Burner
• Sterile DNase RNase free microfugue tubes 2.0ml with o-ring, (e.g., VWR Cat #; 89004-310)
• Tube Rack
• Permanent Fine Tip Marking pen
• Plastic Beakers/Cups
• Lighter or matches
• Gloves

RNA DNA Shield Guidelines

RNA/DNA Shield

• Use RNA/DNA Sheild Solution with fresh tissue
• Before immersion in Sheild Solution, cut tissue samples to ≤0.5 cm in any single dimension.
• Place the fresh tissue in shield following manufacturer’s instructions for the ratio of tissue to Shield.

Sample Collection

Labeling Guidelines

• Country Code
• Timepoint
• Treatment
• Tank

Tool Sterilzation

• Make 10% bleach solution in DI water in plastic beaker labeled #1 10% Bleach
• Pour 100% DI water in plastic beaker labeled #2 DI Water
• Pour 100% Ethanol soluion in plastic beaker labeled #3 100% Ethanol

• Line up Beakers 1, 2, and 3 next to the ethanol burner filled with 100% ethanol

• First, dip the clippers and forceps in #1 10% Bleach
• Second, rinse clippers and forceps in #2 DI Water
• Third, rinse the clippers and forceps in #3 100% Ethanol
• Fourth, sterilze tools by burning off the ethanol
• Fifth, place tools upside down in tube rack with sterile end up

Glove Cleaning

• Make a 5 gallon bucket with 10% bleach
• Make a 5 gallon bucket with fresh water
• Dunk gloved hands in 10% bleach and then fresh water bucket between each sample

Sample Collection Metabolomics

• Label each cryovial tube
• Pick up sample with clean glove
• Cut muscle tissue section to 50-100mg, need to be consistent with the mass you choose so they are standardized to minimize variability in results
• place samples in liquid nitrogen, -80°C freezer, or ethanol cooled with dry ice to snap freeze the samples
• Repeat sample collection process for a duplicate tube for every sample as possible

Sample Preservation for Metabolomics

• Place samples at -80° and store without thawing
  or
• Lyophilize samples and store X

Sample Collection for RNA and DNA

• Fill each tube with desired volume of RNA DNA Shield solution
• Pick up sample with clean glove
• Cut muscle tissue section ≤0.5 cm in any single dimension to obtain the mass you choose
• If you have extra tissue do the same thing for gill, mantle, digestive glad
• Place tissue section in prelabeled 2ml tube with Shield solution as a ratio to your mass
• Repeat sample collection process for a duplicate tube for every sample as possible

Sample Preservation for RNA and DNA

• Store and/or transport samples as follows in DNA/RNA Shield for later purification of high-quality DNA and/or RNA

Temperature	Time
4°C - 25°C (ambient)	Up to 30 days
37°C	Up to 7 days
-20°C and below	>1 year

Sample Collection for Histology

tubes PFA Ethanol

Sample Preservation for Histology

Store in PFA

Sample Collection for Shell

• May want to separate the shell before and after OA?
• Shell Rinsing with DI water?
• Drying at X° for X time
• Store dry and in the darkness if labeled for Calcein

Sample Preservation for Shell