Fish

Lethal and non-lethal tissue sampling in fish for RNA-seq

Written by Juntao Hu on November 2017

Note

  1. To avoid cross contamination, always change equipment (e.g., knife, forceps, razor) before sampling a different individual

  2. Wear mask and frequently change glove to avoid RNA being degraded by RNase

  3. Typical tissue collected from lethally sampled fish: liver, brain, heart, head kidney, skin, muscle

  4. Typical tissue collected from non-lethally sampled fish: gill, fin, blood cell, muscle, scale

  5. Minimum tissue typically required for RNA extraction: 30mg

Lethal tissue sampling

  1. Euthanize fish with a lethal dose of MS-222 (300 mg/L buffered with NaHCO3 to pH 7.0-7.5).

  2. Perform standard dissection procedures to remove aimed tissue.

    1. If in lab, snap frozen tissue using liquid nitrogen and stored at -80°C until analysis.

    2. If no access to liquid nitrogen (e.g., in field), first cut tissue into slices less than 0.5 cm thick as quickly as possible, and then fully submerge the tissue piece(s) into RNAlater (Qiagen) in a tube. Store the tissue submerged in RNAlater for up to 4 weeks at 2-8°C, up to 7 days at 15-25°C, or up to 1 day at 37°C.

    • For archival storage at -20°C, first incubate the tissue overnight in the reagent at 2-8°C. Then transfer the tissue, in the reagent, to -20°C for storage.``

    • For archival storage at -80°C, first incubate the tissue overnight in the reagent at 2-8°C. Then remove the tissue from the reagent, and transfer it to -80°C for storage.``

For non-lethal tissue sampling

  1. Anaesthetize fish with a non-lethal dose of MS-222 (100 mg/L buffered with NaHCO3 to pH 7.0-7.5).

    1. If using blood cell, removing ~3 mL of blood from the caudal vessel using a vacutainer syringe (3.8 cm [1.5 inches], 21 gauge). Immediately centrifuged blood sample for 7 min at 7000 ×g. Following step 3a above for storage.

    2. If using gill, removing <4 mm from the tips of 6 to 8 filaments (0.3 g) from the first gill arch.

    3. If using muscle, gently remove scales from the area on one side of the fish using a knife. Obtain the muscle sample from the dorsal musculature beneath the anterior dorsal fin with a 5-mm (diameter) disposable biopsy punch. Treat wound with Fish Bandage and Betadine antisepic solution.

    4. If using fin, cut off caudal fin near base, and the right pectoral fin (if needed). Put them into a clean tube.

  3. Store samples following the step 3a or 3b above.

Reference

  1. Henderson, C.J. et al. 2016. Assessing the suitability of a non-lethal biopsy punch for sampling fish muscle tissue. Fish Physiology and Biochemistry 42, 1521-1526.

  2. Ackerson, J.R. et al. 2014. Implementation of a non-lethal biopsy punch monitoring program for mercury in smallmouth bass, Micropterus dolomieu Lacepede, from the eleven point river, Missouri. Bulletin of Environmental Contamination and Toxicology 92, 125-131.

  3. Cooke, S.J. et al. 2006. Mechanistic basis of individual mortality in Pacific salmon during spawning migrations. Ecology 8, 1575-1586.

  4. Jeffries, K.M. Consequences of high temperatures and premature mortality on the transcriptome and blood physiology of wild adult sockeye salmon (Oncorhynchus nerka). Ecology and Evolution 2, 1747-1764.

  5. Evans, T.G. et al. 2011. Transcriptomics of environmental acclimatization and survival in wild adult Pacific sockeye salmon (Oncorhynchus nerka) during spawning migration. Molecular Ecology 20, 4472-4489.

  6. Qiagen RNAlater RNA stabilization regent hand book. https://www.qiagen.com/us/resources/resourcedetail?id=38566945-e96b-46a4-a287-ec026849da4b&lang=en