Total RNA isolation for RT-PCR - (Jun/13/2008 )
Hello yall, I have a question for those of you experienced in RNA isolation work. I am trying to get some RNA for downstream RT PCR, but it will be coming from intestinal tissues extracted from mice. I really like the idea of using Trizol because it's got a lot of flexibility (I can increase the volume of reagent if I decide to use more tissue). The only problem is that when it asks you to homogenize tissues, it only mentions things like the Tissumizer or Glass-Teflon, both of which I don't have.
Has anyone tried to homogenize Trizol'd tissues using the QIAshredder? Does that even work?
I've also looked into using the RNeasy kit, as well as the Microfasttrack 2.0 kit (this is more expensive for reasons that I am unsure of). If anyone has other suggestions on good kits to use for RNA isolation, feel free to post away!
No Qiashredder cannot homogenize your tissue. you have to use some kind of mechanical method.
I am using RNeazy protect cell mini kit + QIA shredder for total RNA extraction. I've got very good results and high yield. The electrophoresis of the RNA was also very good. it is a bit expensive because it contains mini columns both for gDNA elimination and RNA extraction, but I think it worth using.
you can try to freeze the tissue with liquid nitrogen and then grind it with a mortar and pestle to powder.