Hi everyone,
I was just wondering if anyone knows the minimum number of embryos one would need to extract mRNA and do a reliable qPCR. I would like to do a relatively stage specific qPCR so I’ll be picking the embryos by hand.
Thanks in advance for any information.
A student in the lab says that 1000 is the minimum, but you won’t really see a pellet or be able to quantify the RNA. For quantifiable amounts, she uses 5000 embryos.
Good luck!
Steve
I have been doing RNA isolation and RNA-Seq, and I’ve gotten enough RNA to do that with as few as 200 embryos. I would suggest for small amounts of RNA to use something like the Qiagen RNeasy Micro kits. When you are working with very small amounts of RNA, the pellet is either so small you can’t see it, or it will have trouble precipitating even with a carrier like glycogen. Using Trizol LS for the initial RNA isolation, and running the aqueuous layer through and RNeasy micro column, I average about 25ng of total RNA per 200 embryos. There are a number of protocols for this online. Good luck!
With the new available techniques, one can be enough. (depending on the stage you are looking at and the putative number of cells, which express your gene of interest)
If you look at this paper : http://www.ncbi.nlm.nih.gov/pubmed/?term=cel-seqn they are doing single blastomere RNA-seq.
Because you linearly amplify your RNA it is reliable for quantifications. If you can do RNA-seq, you will certainly be able to do qPCR.
Of course it will be important to set your conditions first but I really think that this method will be perfect if you have a small amount of embryos.
PS: I attend to a talk of Itai Yanai, and they are doing some sort of RNA-seq time course of embryo development, and I really doubt they are usind 1000 of them for each stages.
I hope that my answer