I used Isolate (similar to percoll) to separate semen into mature and immature spermatozoa. However, if you need all spermatozoa independent of maturation status, you can centrifuge semen and then wash the pellet with cell culture medium to get rid of the seminal plasma. As the semen sample is not divided by its density, different cells (not only spermatozoa) will be located in the pellet.

For RNA isolation use Trizol or TriFast according to the manufacturer. You can store the sperm pellet or the trizol lysed sample at -80 C for at least a month.

There is also possible isolate the spermatozoa from other cells on the base of their motility. I suppose:

1.       dilute the semen at least 1:5 using the phosphate buffered saline

2.       centrifuge

3.       remove the liquid

4.       overlay the remaining pellet gently by 1 ml of the phosphate buffered saline

5.       let the spermatozoa migrate into the liquid phase cca 30 minutes

6.       move the liquid phase containing motile spermatozoa into new clean test tube

7.       centrifuge again

Best regards

Pavel

I know I am coming into this very late in the day but would be interested in additional perspectives. I have had many conflicting conversations. We are currently freezing sperm pellets (washed in PBS post swim-up, then stored as pellet). While this will be fine for DNA extraction, I have some concern about small RNA integrity. The option we have is immediate small RNA isolation and freezing at -80C but this also has issues as samples trickle in (it will take up to 12 months for collection), or what we currently are doing which is freezing the sperm pellet (snap frozen and stored at -80C). Alternatively we could use RNAlater. Our aims are to analyse both DNA methylation and small RNAs. We have plenty of samples. It is human sperm. Thank you.