Hi Hildegunn,

I have got many years of experience in RNA isolation. For forzen tissues I really advise the RNAlater-ICE (blue colored liquid). Here you can find furter details: https://www.thermofisher.com/order/catalog/product/AM7030.

I have used it earlier for snapfrozen mice brain tissue RNA isolation, with success.

Or for financial purposes, you can you use the more cheaper good old acidic phenol-guanidin-isothyocianate mixture (e.g. Trizol, TRI Reagent for comm. use) for lysis.

Both of the upper lysis solution adding steps further followed by homogenization by mechanical method (stainless steel blades homogenizator or beads). Then use in classical phenol-chloroform-isopropanol purifing method. (Pros: the cheapest, intact total RNA (including rRNAs, miRNAs) can isolated with high yield, Con: toxic organic solvents (need a chemical hood), very problematic to use in high throughput).

You can use the Trizol lysate directly in Direct-zol RNA spin column kit (https://www.zymoresearch.com/media/amasty/amfile/attach/_R2060_R2061_R2062_R2063_Direct-zol_RNA_MicroPrep_ver._1.0.0.pdf). I have very good experiences with this kit in human chordoma tissue RNA isolation in the terms of RNA quality and quantity. I have used the Trizole lysate after chloroform phase-separation step in the above kit. To decrease the protein contamination in the further purification steps

If you have any questions about RNA isolation from human blood, tissues samples I will answer, if I can.

Bests,

Tamas

I think this paper is good for you:

Das PJ, Paria N, Gustafson-Seabury A, Vishnoi M, Chaki SP, Love CC, Varner DD, Chowdhary BP, Raudsepp T. Total RNA isolation from stallion sperm and testis biopsies. Theriogenology. 2010 Oct 1;74(6):1099-106.

Thank you Tamas, for your insights! I will for sure look into the RNAlater-ICE.

Zahra - Thank you for the nice article!! It looks just up my lane - although working with stallion sperm gives you the luxury of plenty sperms to work with. :)

My biggest worry working with mice miRNA is contamination of somatic cells (containing ~600folds more RNA than sperm), loss of sperm cells in washing steps, and of course RNA degradation as time from thawing to lysis step takes (in my initial protocal) app 60 min (I need to reduce this time).

Best,

Hi Hildegunn,

When using RNAlater-ICE it is reversible block the RNases until the sample in the solution. After removal the reversible effect can decrease by elapsed time.

To tell the truth I don't know your purification protocol. I haven't done such protocol before. Maybe if it is possible to use sperm cell specific cell surface marker to isolate these cells by magnetic bead connected anti-sperm cell surface marker antibody. But it can be more expensive (because of the antibody (if available), and magnet system to catch the magnetic beads.) But I'm sure that it can be more clear than other chemical isolation methods. It was just an idea.

Have a nice day.

Tamas

Hi Hildegunn,

Did you figure it out how to extract RNA from frozen sperm?

I am trying to find a good protocol for sperm purification from frozen epididymis. I do not have time to extract sperm from fresh tissue and my tissue will come from another city, so it needs to be frozen. If you have some insights, please let me know.

Nayara Lopes

I`m sending you a PM :)

https://www.thermofisher.com/order/catalog/product/AM7030.

Ali Olfati

Thank you for this tip! :)