I am trying to isolate RNA from sperm using Trizol, but I can not get good results. I have some protocols to give a try and they suggest keep the sample + Trizol on ice. However, I had seen some protocols suggesting put the sample + Trizol in a heat block at 65C for 15 min. My question is: what is the difference between heat and ice for RNA isolation? How about RNA integrity?
What do you mean with "can not get good results"? Sample degradation, low recovery...? Heating samples in trizol is quite unconventional and risky. Have you tried some extraction kits?
Hello Andrea, regarding your question, for me good results means high amount of isolated RNA. And yes, I had tried RNeasy mini kit from qiagen, but I can not use very high number of cells, so I am testing the trizol protocol. Some researchers had been used heat for RNA extraction (Ostermeier et al, 2002 and 2004; and Feungang et al 2010). But I don't know how this step helps to isolate RNA.....
In order to improve the yield of RNA recovered from a few cells, we are very happy with a commercial kit from Norgen biotech. I recommend you to try it, it is cheaper but more efficient than Qiagen. You can read our publications (I can let you have the final PDF if you want) about a comparison of these kits:
Article Quantification of Small Non-Coding RNAs Allows an Accurate C...
A book chapter
Chapter Quantification of Small Non Coding RNAs May Allow Accurate C...
Here are the links: http://dl.dropbox.com/u/1397051/Papers/JBB-659028%20-%20miRNA.pdf and http://dl.dropbox.com/u/1397051/Papers/JBB-659028%20-%20miRNA.pdf.
Hope they work! Let me know
Thank you guys, I appreciate the replies... I will think about it.
Once the Trizol extraction is separated into the phases, I can't imagine temperature affecting the overall concentration/quality of the RNA. Not to muddle the protocol more, but I perform the Trizol separation/extraction at room temperature, and found that centrifugation for the phase separation at 4 C did not affect yield or quality.
Perhaps your samples are more difficult to 'dissolve' than typical tissue? In that case, extra pipetting until the Trizol-cell solution is no longer viscous maybe required. The only other advice I have is to take extra care with pipetting the aqueous phase, and to use carrier Glycogen during the EtOH precipitation to avoid loss of the pellet, since there will be a limited amount of RNA from a relatively small number of cells.
Best of luck!
Hi Aletheia, do you use any RNA isolation kit? I think it should have Trizol protocol.
Hi Ahmet, yes I usually use RNeasy mini kit plus from Qiagen, but it is so expensive over here that I am trying to move on to Trizol. However, none of protocols I have tested worked well on my samples. I did not try glycogen yet, and I think my samples membrane are really hard to disrupt. For this I performed needle (at least 10 times), pistol and vortex (high speed) for 5min. Nothing changed....Glycogen will be my last shoot. Thanks for all responses :-)
Aletheia, I would not vortex your samples. This causes extra shearing that, quite frankly, is not necessary. If you add Trizol to your cell pellet and pipette up and down until the solution is no longer viscous, then you will surely have 'dissolved' all the cells into the highly reactive Trizol. Then by adding chloroform and hand shaking the vial 5-10 times, you can start the separation process, with as little shearing force placed on your RNA as possible. Hope this helps!
Certainly it helps, but I realized that my samples get sticky when I add Trizol...
I am stuck on this protocol for a long time, so I will include glycogen on my current protocol, and give a try for the last time. Thanks Christopher for your advices.
Just in case another protocol would be helpful:
1.) Aspirate supernatant from pellet.
2.) Add 1 ml of Trizol reagent to each pellet. Invert several times.
3.) Pipette homogenate up and down until reagent is no longer viscous. Transfer homogenate to a microfuge tube. Incubate in the hood for 5 min.
4.) Add 0.2 ml chloroform to homogenate and shake for 15 seconds. Incubate in the hood for 2 minutes.
5.) Centrifuge at 150000 * g for 15 minutes.
6.) While centrifuging, add 0.5 ml isopropanol (100%) to a new microfuge tube. Add 10-20 ug of RNAse free glycogen to the isopropanol.
7.) Transfer 0.5 ml of the upper aqueous layer (containing RNA) to isoproanol tube.
8.) Store at -80 C to fully precipitate RNA/Glycogen.
Thanks a lot Christopher, I really appreciate it....I will test this protocol, and I'll let you know :-)
Aletheia, this my kit's protocol. I highlighted trizol section. Maybe it helps you.
Thanks Ahmet, I will take a look on this protocol. All the best.
I suggest to use stainless beads and QIAZOL for homogenization.
if you still have problems, email me for a full protocol
@shlomit Kenigsberg. Could you please share with me your QiaZol protocol for RNA isolation.
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