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Methods
How To Make Tissue Samples for Molecular Studies
A: Use clean, sharp instruments for taking samples.
B: Take a fin clip or muscle plus from the righthand side of the fish.
C: Place fin clip or muscle plug in an air tight container.
D: Make sure Tissue number and Voucher number match.
F: A cheap yet effective tagging method.

Increasingly molecular tools are being applied in a range of fish biodiversity studies and as a result the value of preserved specimens is greatly augmented by tissue samples taken from voucher specimens. The use of PCR techniques to amplify DNA means that only a small amount of tissue need be taken in order to provide an adequate amount of DNA for analysis. Tissue samples should be taken from specimens as soon after death as possible as this results in the least degraded DNA samples.

AVOID CONTAMINATION:
When taking tissues the most important precaution is to avoid contaminating the sample, either with your own DNA or with that of another specimen. To reduce the risk of contamination all sampling should be made with clean, sharp tools, and all tissues handled only with forceps - not fingers. After sampling one specimen and before sampling another rinse the forceps, scalpel blade and/or scissors in clean water and to dry them with a clean paper towel. Most importantly, fish specimens must not come in contact with formalin prior to tissue sampling.


TISSUE SAMPLING:
For most standard DNA sequencing applications tissue from just about anywhere on the fish will suffice. However, tissue taken from a readily accessible location and which requires minimal dissection is best as this will result in the most intact voucher specimen.

FIN CLIPS:Clips from the paired fins are a good choice, and should always be taken from the right hand side of the specimen. These can easily be removed by holding the fin erect with forceps and sniping off the body of the fin with a sharp pair of scissor. An alternative is to remove the lower lobe of the caudal fin. This is often useful when sampling very small specimens, however it is more destructive than removing one of a pair of fins. MUSCLE PLUG: Another option is to remove a small plug of muscle tissue from the right hand side of the caudal peduncle (tail). In scaled fish scrape away a few scales and cut three sides of a rectangle with a sharp scalpel blade, then slide the blade parallel with the vertebral column and remove the rectangular plug of muscle either leaving a flap of skin attached or remove the skin as well.

STORAGE:
Carefully place the fin clip or tissue plug into a screw top vial or if that is not available then any clean, airtight tube or bag will do. Cover the tissue with 95% ethanol and seal the container. Be sure to include a small alcohol proof label with a unique tissue number (written in pencil) inside the vial so that the tissue can be linked to the voucher specimen from which it was taken. Using an indelible marker also write the tissue number on the outside of the vial and on the lid. The fish voucher specimen is now ready to be fixed in 10% formalin solution.

If no alcohol is available, a back-up option is to remove a fin clip or small muscle plug and allow it to air dry on a clean paper towel. The dried tissue sample can then be placed in a vial or sealed plastic bag with a tissue label and stored.

Alcohol filled vials and dry tissue specimens are best stored at a cool temperature and the sooner DNA extraction can take place the better.

TAGGING VOUCHER SPECIMENS: Once a tissue sample has been taken it is important that that tissue can readily be linked with its voucher specimen. To do this we employ a cheap but effective method using a Dymo letter press and a garment Tag Gun. For each tissue voucher an individual Dymo tag tissue number is made (this number corresponds to the numbers on and in each tissue vial) and the needle of the tag gun is passed through that label (Fig. FA). The needle is then passed through the lateral muscles of the voucher specimen just above the anal fin (Fig. FB). The specimen is then tagged with a permanent label (Fig. FC) that ties it directly to the corresponding tissue sample. This is a cheap and efficient way to individually tag tissue specimen vouchers.





NOTE: A useful manual outling more details on DNA Sampling Protocols is available here.

© 2007 American Museum of Natural History Back to Ichthyology Department