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RNA extraction from human sperm

RNA extraction human sperm

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#1 Sunflower_gl

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Posted 12 September 2011 - 02:10 AM

Hi every one,
I'm a first year PhD student. My research forcus on the male infertility and gene expression during the spermatogenesis. I've extracted RNA from human sperm. But my samples will come from an other country, normally they will be send by post and certaintly it take at leats 3 days. I don't know how can I do for preserving my RNA spermatozoal from the degadation because they are very sensitive.
If you know some method for preserving RNA, help me, please!
Thanks so much,

Sunflower_gl

#2 bob1

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Posted 12 September 2011 - 03:25 PM

I am a little unsure if you mean that the sperm are coming from another country or just the RNA

If you can get the RNA as a dried pellet, that should be fine for a while at room temperature (provided it is dry enough to prevent RNase activity).

You are best off shipping RNA on dry ice if you can.

For the sperm, ideally you could transport it frozen on dry ice, much as they would for sperm used in IVF, then thaw, spin down and proceed from there. Otherwise you could get them to send it dissolved in trizol or possibly in RNAlater (check the manual for this product).

#3 Sunflower_gl

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Posted 13 September 2011 - 12:56 AM

Hi,
Sorry about the unclear infos. I mean the sperm will be come from another country :)

I've found some interesting informations on the Quiagen's site. In fact, I can use the RNA protect cell reagen kit. The sperm pellet would be dissolved in this product and then it could be stored at room temperature up to 7 days. Well, it would be fine. I'll try it and let see how it works ;)

Thanks for your help.

#4 Sunflower_gl

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Posted 29 September 2011 - 11:55 PM

Hi, my RNA were isolated from these solutions using a RNeasy Kit, Qiagen, according to producers's manuals with some modifications. So, I've added 10µl 2-ME to 1ml RLT, but the quantity of RNA obtained was less and the quality of RNA evaluated by Nanophotometer was worse in comparison with the RNA extracted by RLT only.
I knew that 2-ME can eliminate ribonuclease released during cell lysis, so it protect our RNA during RNA isolation procedure. But I don't understand my results.
Does someone have the same problem with RNA using RLT added 2-ME?
Best wishes





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