I am taking peripheral blood and enriching for Neutrophils (using Ficoll+ NH4cl. I also tried Ficoll+dextran, as well as Ficoll+hypotonic treatment). In all cases I am getting satisfactory enrichment (measured in Heamatology cell analyser, CBC count). Then I went on to isolate the total RNA from the neutrophil enriched pellet using a Trizol+Ambion kit. Once I checked in bioanalyser the RNA seems to be degraged. I don't understand what is going wrong. 10 ml blood, so there are heaps of cells. Any ideas of advice?
Are you isolating neutrophils,lymphocytes or total WBCs?
You need to use ficoll if you want PBMCs(lymphocytes & monocytes)Check your NH4Cl concn for RBC lysis,use RNAse free reagents..check RNA concn using nanodrop/spectro whether it is tallying with inc no.of cells.It seems your RNA getting degraded durimg cell lysis.You can include RNA Later reagent also.
I am isolating Neutrophils. I checked in Nanodrop and also in bioanalyser too. It doesnt tally up. I think too the cells getting lysed. But I have to isolate Neutrophil anyways. In that case how Will I get around that. And also if I use RNA later at which stage you think I should use it. To the blood, so it stop all the reactions or after getting the neutrophil pellet.
I think it is happening due to some simple reason.There are 2 different aspects,either the cells get lysed during isolation or the RNA get degraded too early.It is better to look in to your buffers (pH/salts etc.)and NH4Cl concentration.We regularly used to isolate RNA from total WBC without density gradient and without using RNA Later for expression assays without any problem.
Thanks for your comments. I made some modification to the protocol, will give it a go soon. What could be the reasons for early degradation you think.
Presence of RNAse either from lysed cells or reagents/water is not RNAse free..can be a likely cause I presume.
Hello,
it is known that neutrophils contain comparatively small amounts of RNA. This means you need at least PMNL from 10 mL blood to get reproducible results (own experience). As it was posted before, the cells should be processed as quick as possible, the half life of a neutrophil derived from a healthy person is about 7-8 hours. Also important to consider is the risk of contamination with other leukocytes especially with monocytes. Monocytes contain roughly 20 fold more RNA than neutrophils so there is a high risk of bias in your subsequent analysis in case of monocyte contamination.
We also have good experience with polymorphprep but this medium has ist limitations. If the blood donor suffers from severe systemic infections the efficacy of polymorphprep drops and the risk for contamination increases.
Best wishes for your Research
Dirk