whole blood storage for RNA isolation - (Dec/22/2008 )
Hey all, i am here again.
Truly speaking, i have none experience in dealing blood to isolate the RNA with neither any RNA isolation method. Hope there is a reply from any of you in this post.
Like my previous two posts: http://www.protocol-online.org/forums/inde...showtopic=41894, http://www.protocol-online.org/forums/inde...showtopic=41764
I have a problem of store the whole blood for RNA isolation since i am not going to isolate the RNA immediately.
I read some information that Tri-reagent is specifically used to isolate RNA from whole blood. However, i have some questions in mind:
1. The Tri-reagent can be used to store the whole blood? how about the buffy coat fraction?
2. The point of blood collection doesn't have an equipped molecular lab, with no aspirator, and no sterile workbench. Can i separate the buffy coat (1,000-1,500 x g, 10 min) from the whole blood, and pipette out (with no aspirator help) to collect the buffy coat only?
3. what are the maximum period can store the tri-reagent contained whole blood/buffy coat in -80c to have a good RNA quality?
Need ur help. Thanks first. =)
Truly speaking, i have none experience in dealing blood to isolate the RNA with neither any RNA isolation method. Hope there is a reply from any of you in this post.
Like my previous two posts: http://www.protocol-online.org/forums/inde...showtopic=41894, http://www.protocol-online.org/forums/inde...showtopic=41764
I have a problem of store the whole blood for RNA isolation since i am not going to isolate the RNA immediately.
I read some information that Tri-reagent is specifically used to isolate RNA from whole blood. However, i have some questions in mind:
1. The Tri-reagent can be used to store the whole blood? how about the buffy coat fraction?
2. The point of blood collection doesn't have an equipped molecular lab, with no aspirator, and no sterile workbench. Can i separate the buffy coat (1,000-1,500 x g, 10 min) from the whole blood, and pipette out (with no aspirator help) to collect the buffy coat only?
3. what are the maximum period can store the tri-reagent contained whole blood/buffy coat in -80c to have a good RNA quality?
Need ur help. Thanks first. =)
Hi,
My experience with this kind of thing is limited but I have extracted RNA from parasite infected RBCs with Trizol and stored at -80 for a week or so before shipping to another lab on dry ice. The RNA must have been ok because they got a publication out of it.
My lab also did not have an aspirator and I managed to separate plasma, buffy coat and RBCs with a pipette. Are you worried about contamination of your samples?? Just find a clean area of bench, wash down with RNAse-ZAP or 70% ethanol and work under a bunsen flame - not ideal but it might be ok.
Hope this helps a bit,
P
Truly speaking, i have none experience in dealing blood to isolate the RNA with neither any RNA isolation method. Hope there is a reply from any of you in this post.
Like my previous two posts: http://www.protocol-online.org/forums/inde...showtopic=41894, http://www.protocol-online.org/forums/inde...showtopic=41764
I have a problem of store the whole blood for RNA isolation since i am not going to isolate the RNA immediately.
I read some information that Tri-reagent is specifically used to isolate RNA from whole blood. However, i have some questions in mind:
1. The Tri-reagent can be used to store the whole blood? how about the buffy coat fraction?
2. The point of blood collection doesn't have an equipped molecular lab, with no aspirator, and no sterile workbench. Can i separate the buffy coat (1,000-1,500 x g, 10 min) from the whole blood, and pipette out (with no aspirator help) to collect the buffy coat only?
3. what are the maximum period can store the tri-reagent contained whole blood/buffy coat in -80c to have a good RNA quality?
Need ur help. Thanks first. =)
Hi,
My experience with this kind of thing is limited but I have extracted RNA from parasite infected RBCs with Trizol and stored at -80 for a week or so before shipping to another lab on dry ice. The RNA must have been ok because they got a publication out of it.
My lab also did not have an aspirator and I managed to separate plasma, buffy coat and RBCs with a pipette. Are you worried about contamination of your samples?? Just find a clean area of bench, wash down with RNAse-ZAP or 70% ethanol and work under a bunsen flame - not ideal but it might be ok.
Hope this helps a bit,
P
Hi
thanks for the comment.
will it get more tricky if i use the buffy coat to isolate the RNA by trizol method? i couldn't find any detail of store buffy coat for RNA analysis or to isolate the RNA from buffy coat using trizol/tri-reagent BD method.
Hi again,
I just did a quick search and found this paper
Int J Epidemiol. 2008 Apr;37 Suppl 1:i11-5.
Levels of 5' RNA tags in plasma and buffy coat from EDTA blood increase with time.
Salway F, Day PJ, Ollier WE, Peakman TC.
PMID: 18381387
The methods say they collected the buffy coat and plasma and stored them directly at -80C doing the Trizol extraction at a later date.
P