RNA purification from macrophages - (Jul/25/2005 )

I have a problem in purifing RNA from macrophages.
I am using RNeasy of Qiagene and versagene of gentra and in both kits i got very low yield of RNA around 3ug from 5 million cells.
Do anyone tried to purify RNA from macrphages and what yield did they get? or maybe th problem in the kits?

hi
a colleague who extracts RNA from macrophages use phenol based method "Tri" family (Tri zol and Tri reagent...) and get up to 30-40 µg from half a 15cm plate at 70% confluency. They do not use a specific kit or columns...
Sorry for not helping you more...
fred

hi
yes they're human macrophages. Due to the fact its for siRNA detection they do not use any column based kit. Btw, i've noticed that tri family are not that unconfortable, and cheaper as kits. They started from 5.10^6 macrophages and yield about 100µg RNA.
Honneslty i prefer tri family because if you get more cells you can increase the in lysis step the amount of "tri" used, and gets better lysis as in kits.
Hope that helps you.
Fred

ps : They're currently in holidays (yeah some people have ones ) so i can ask them when they get back...

hi
yes they're human macrophages. Due to the fact its for siRNA detection they do not use any column based kit. Btw, i've noticed that tri family are not that unconfortable, and cheaper as kits. They started from 5.10^6 macrophages and yield about 100µg RNA.
Honneslty i prefer tri family because if you get more cells you can increase the in lysis step the amount of "tri" used, and gets better lysis as in kits.
Hope that helps you.
Fred

ps : They're currently in holidays (yeah some people have ones ) so i can ask them when they get back...

hi
for general advice, double the amount of TRI recommended in the protocol. Efficiency of lysis is greater and yield too. I use a p1000 to solubilise and homogeneize the cells until the solution becomes clear.
I heat the lysis solution to 50° before using it. Accelerates the lysis proccess and efficiency...