Hello, I need to extract DNA and RNA from fish mature spermatozoa and unfertilised oocytes. I am also looking for a protocol to isolate mature spermatozoa from fish semen/testis and any advice on how to proceed with oocyte DNA/RNA extraction.
Dear Lucrezia,
Here at our institute we have extracted DNA from eggs for paternity evaluations, and RNA for gene expression. The persons that can help you with the protocol are
Dr Costas Tsigenopoulos and Dr Elena Sarropoulou.
Please contact me on my email ([email protected]) to make the connection with the above researchers.
Best regards, Dinos
Dear Lucrezia,
I have attached my methods for DNA and RNA extraction from lymphocytes, which of course are diploid cells. It may require some adaptions but basically the principles should be similar, as you basically need to lyse the appropriate membranes (cell and nucleic) to isolate different parts of the cell. The methods are based on the fact that DNA is confined to the nucleus and RNA is found in the cytosol. The nuclei of the lymphocytes extracted from peripheral blood remain in tact. If this is the same for sperm and oocytes then the same method can be used but may need adaptions for haploid cells. The RNA isolation from the cytosol is very similar to any other nucleotide extraction.
Hope this is helpful.
Best wishes, Annwyne
We regularly genotype fish sperm. We release it, spin it down gently, and from there we treat it just like a piece of tissue with a typical SDS genomic DNA extraction buffer and a normal phenol:chloroform extraction. Let me know if you want details!
Oocytes obviously have far less DNA, I doubt the same method would work there.
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