RNA ectraction from whole blood using TRIzol LS - (May/17/2006 )
Hi! I am trying to isolate RNA from whole blood using TRIzol LS(My samples are preserved in EDTA.
) I have tried several times but do not get enough RNA(I need at least 3μg) and have also experienced problems with DNA contamination.The isolated RNA will be then used to perform RT-PCR. Any suggestions?
How much blood are you using? I would suggest if you are using less than 5ml you may not get the yield you require
use more trizol (maybe you're too short in trizol and this fact doesn't allow to get the required acidic pH during pase separation phase, which drives a DNA contamination).
Use glycogen or an other carrier during isolation. Note than you need to add the carrier before adding isopropanol.
So far I have tried out two different protocols using 100ul and 150ul of whole human blood.But since I could not get enough RNA I used 500ul of blood, added 500ul of RNase free water, incubated in ice, centrifuged for 25min at 2500rpm,redissolved the pellet in RNase free water and followed the next steps of the protocols.I understand that I may need more blood but the patients are children and most of the blood samples consist of maximum 1ml(sometimes even less). I am also trying to isolate RNA from bone marrow samples which of course consist of less than 0.5ml.How can I get enough RNA (minimum 2ug) from such small quantities of blood samples?
I understand that low quantity of TRIzol LS could be held responsible for the DNA contamination and I am currently working on fixing that .
Thanks!
Use glycogen or an other carrier during isolation. Note than you need to add the carrier before adding isopropanol.
You are probably right.I have also concluded that I am not using enough TRIzol.I am now trying to fix that.Any ideas on how to isolate enough RNA (minimum 2ug) from less than 0.5ml of blood?
Thanks a lot!
well i'm not too sure of that, but what is the goal of the incubation of RBC in water ? Is it for lysis purpose? why don't you directly lyse in trizol ? You'll quite surely reduce RNases effects, getting probably more RNA
For increasing RNA global precipitation, you may use a carrier as glycogen as i said in previous post.
For increasing RNA global precipitation, you may use a carrier as glycogen as i said in previous post.
Hi Fred!
Since the laboratory is a bit short in equipment right now and I have to use eppendorf 1.5ml for the extraction, the purpose of the incubation was to actually reduce the volume of the sample and still get as many WBC as possible.However I didn't realise that this would lead to RNA degradation...Any ideas on how to make it work without previous RBC lysis starting with maximum 250ul of sample?
Thanks for the tips and for putting all this thought in this matter!