Total RNA from lymphocytes - Problems with RNA yield (Jul/07/2005 )
I am trying to extract RNA from initially lymphocytes and eventually just B-cells.
I have been using ~30mls of whole blood and separating the lymphocytes by using Ficoll.
After washing and counting I have been using the Promega SV kit to isolate RNA but the yields seem to be low.
Is anyone else doing this and what sort of yield should i expect??
I am confident the kit is okay as i have used it many times before for cell lines which yield many micrograms of RNA, so not sure where i am going wrong.
Though i am using bacteria for RNA isolations i have found a standard 10ml culture to be useless for RNA extraction i have had to use 50ml cultures to ensure a good isolation, you could try using 60ml of blood it may help.
I personally don't have much confidence in Promega's RNA isolation kits. Try one from Ambion or Qiagen. 30ml seems like a generous plenty amount of starting material to get a decent amount of RNA from.