Extraction of RNA from blood samples - Problems (Trizol method) (Apr/23/2010 )
I have a problems extracting RNA from Trizol samples that I have been given for a study and I hope someone can help. I received the samples in Trizol so I will do my best to my knowledge to describe the method that my colleagues follow in their lab. Blood is collected and is cultured over night with a stimulant. The blood is then harvested using the Ficoll plaque method to isolate the lymphocytes. This involves taking the buffy coat layer from the solution. The samples I have obtained appear to have more than trizol present and I think perhaps some of the Ficoll plaque buffer has also been taken over in to the trizol as there appears to be an aqueous solution present. As you can imagine this generates a problem when you add the chloroform as there is already an aqueous layer present. I don't really want to remove the aqueous layer that is already present in fear that the RNA is in this layer but it is interfering with the extraction and in some samples I am getting a thick powder pellet which I assume is salts from the buffer.
As these samples are precious I wondered if you have ever encountered this problem and if you know of a method to possibly remove the salts from the sample to leave only the RNA. I was wondering if the use of a column or something could possibly work?
Your help on this matter would be much appreciated.
If your blood specimens were frozen whole blood, AquaPreserve could help and extract total blood RNA, but I don't know if it would work with Trizol-blood. If you would like to give it a shot at mixing x volumes of AquaPreserve to 1 vol of Trizol-blood, please let me know.