Hi!!!I'm currently trying to isolate RNA from mice tails (with the NucleoSpin kit from Macherey-Nagel) in order to genotype them. But the concentration I measure in nanodrop is 5-9 ng/ml and even after amplification with PCR I still can't see bands in my agarose gel.Have you got any idea what is the optimal RNA concentration that can be isolated from the tail?And what is the problem with my isolation?Thank you in advance!
I have not any experience of isolating RNA from mice tail but I have isolated RNA from human epithelial tissue using miRVana Kit from life technologies as well as TriZol method. From my experience as well as experiences from my colleagues I can say that yield from TriZol method will be definitely higher than any kit based method. Using liquid nitrogen for crushing tissue samples will also be helpful.
Hi Penelope, just to make sure, why isolating RNA to genotype mice? Typically, isolating genomic DNA is the standard procedure for genotyping... and that's quite simpler and cheaper...
Anyway, RNA isolation is tricky and always requires some training. The problems can be many, the lysis of your tissue (cytoplasmic or total? inefficient? Tail tissue is mainly hair, skin, bone... not an easy tissue to lyse, best is with proteinase K in 1% SDS buffer, but then in such denaturing conditions using the kit is not possible, so phenol/CHCl3 extraction would be the way), the kit itself (make sure you use the NucleoSpin RNA kit... there are many NucleoSpin kits from Macherey-Nagel for different nucleic acid extractions), degradation of the RNA (best is to work on ice or in the cold room and spin at 4 C), the RNA recovery in EtOH (if you are extracting little amounts in high volumes it will also precipitate inefficiently). Indeed 5-9 ng/ml is close to nothing, you need at least 200 ng for a RT reaction if you use qPCR, but 500 ng for end point PCR, of course you will never get any PCR amplification with 50-100 pg. In any case you should have a positive control for your PCR reaction, because that's the last difficult point, PCR reactions need to be optimized, often the primers one designs are not efficient or require tuning the cycling conditions...
Thank you both for your help!Especially the questions and the little-important tips will definitely improve my work.
Hello Penelope. I have the same problem.. could you tell me please if you found out how to isolate RNA from mouse tail?
I use Qiagen RNA isolation kit , but I get low yield. I think the problem is that the mouse tail is very hard to disrupt and homogenise
- Badges
- Science method