Ficoll Hypaque Method

So far, most gene expression analyses of peripheral blood have been carried out on PBMCs isolated from anticoagulated phlebotomy samples,25'294041 because PBMCs are the most transcriptionally active cells in blood along with granulocytes. During phlebotomy, whole blood is usually drawn in standard blood collection tubes, but different anticoagulants, e.g., EDTA, sodium citrate, and heparin, are used. The subsequent separation of PBMC from other blood cells, such as erythrocytes, can be performed via a density-gradient medium (Ficoll-Hypaque).4243 In this method, the anticoagulated blood/buffy coat sample is layered on the Ficoll solution and centrifuged for a short time period. Differential sedimentation in the density gradient medium during centrifugation results in separation of the different cell types. The duration of the whole procedure is about 2 h, depending on the number of samples processed in parallel. The isolated PBMC are then lysed and subjected to RNA isolation, e.g., with TRIzolĀ® Reagent (Molecular Research Center, Inc., Cincinnati, OH, U.S.), a phenol/chloroform guanidine thiocyanate-based isolation method according to Chomczynski.4445 The main advantage of this approach is that it constitutes a relatively inexpensive method for the isolation of lymphocytes and mono-cytes from blood samples. However, in comparison to the QIAamp method, isolation of PBMC by Ficoll-based technique is time-consuming, requires skilled processing, and multiple variants of the procedure exist.

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