An optimized method to separate reticulocytes from peripheral blood for molecular analysis.
A method based on immunomagnetic sorting of reticulocytes from peripheral blood was set up and combined to a commercial extraction kit for the isolation of total RNA from whole blood. This procedure resulted in high-quality RNA samples suitable for molecular analysis. We used this procedure to analyse erythroid-specific transcripts, starting from peripheral blood samples, to search for differently expressed mRNAs in patients with hereditary persistence of foetal haemoglobin. After erythrocyte lysis, CD15(+)and CD45(+) peripheral cells were negatively sorted to remove leucocyte populations that could have affected the subsequent screening procedure. The cell sorting and RNA extraction procedure was completed within 1-2 h of erythrocyte lysis, which represents a consistent saving of time compared with other procedures. Moreover, it produced 1 microg of total RNA per ml of blood samples, which is sufficient for molecular analysis. Therefore, our method is a reliable and efficient tool to isolate RNA from specific cell subpopulations poorly represented in peripheral blood, particularly when accurate detection and characterization of highly unstable and poorly expressed molecules is required.
Petruzzelli
R
R
Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, Naples, Italy.
Palumbo
I
I
Gaudino
S
S
Scalia
G
G
Del Vecchio
L
L
Izzo
P
P
Grosso
M
M
D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D012156:Reticulocytes / Q000502:physiology*
D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human