| | Re: RNA purification from reticulocytes
Adult human reticulocytes are isolated from 20 ml of peripheral blood in heparin following a process of leukodepletion. Leukocytes are concentrated by centrifugation at 3500 rpm for 30 min at 4 °C, forming a disc (buffy coat) at the plasma-red cell interface. Both the plasma and buffy coat are removed, and the cells are washed three times with 10 ml of ice-cold PBS. At the third wash, reticulocytes are concentrated by centrifugation at 3500 rpm for 30 min at 4 °C. The supernatant is removed, and the top 700 μl reticulocyte-rich fraction is added to 4 ml of ice-cold PBS. This red cell suspension is layered on top of a column consisting of about 4.5 ml of 2 parts cellulose (Sigma cod. C8002) and 1 part sigmacell type 50 microcrystallin cellulose (Sigma cod. S5504) in normal saline. The red cells are eluted by gently centrifuging the column at 1000 rpm at 4 °C for 1 min. This leukodepleted eluate (∼2.5 ml) is washed three times in 10 ml of ice-cold PBS. Finally, the reticulocyte-enriched eluted cells are recovered by centrifugation and resuspended in 2 ml of ice-cold PBS. RNA is extracted with 5 ml TRI reagent (Sigma cod. T9424).