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RNA purification from reticulocytes

RNA purification from reticulocytes - RNA Forum

RNA purification from reticulocytes - RNA Forum. Discuss RNA including mRNA structure and function, rRNA, tRNAs, and the biochemistry of other RNA molecules.


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Old 08-01-2011, 02:04 PM
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Default RNA purification from reticulocytes



Hi, does anyone have the protocol to extract RNA from reticulocytes?thanks!!!
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Old 02-12-2013, 03:56 PM
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Old 03-17-2014, 04:04 PM
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Default 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).
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Old 10-02-2014, 11:16 AM
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Default Re: RNA purification from reticulocytes

Reticulocytes manual count and classification Dispense in tube, 100 l of brilliant cresyl blue (0.5% BCB in 0.8% NaCl, 0.7% trisodium citrate in dH2O and filter before to use) and to add 200-250 l of whole EDTA fresh blood samples, mix and incubated at 37 C for 20-25 min. The blood volume to be added to the dye depends on the volume of packed red blood cells volume (PCV), 200 l for PCV equal or superior to 30% and 250 l for less than 30% PVC. At the end of incubation, mix again the contents of the tube well and place a small drop of the mixture on a clean glass slide and prepare a thin smear. Counterstain with Wright's stain, if desired. Allow smear to air-dry and reticulocytes are counted, for up to 72 hours. Place the slide on the microscope stage and, using the low power objective, locate the thin portion of the smear in which the red cells are evenly distributed and are not touching each other. Switch to oil immersion magnification and count the number of reticulocytes in 5 fields of 200 RBCs. For to determine Ret% calculate dividing number reticulocytes counted/10. When Hct value of the sample is different to Hct-normal people, about 45, is need to correct reticulocytes value obtained using the following formula : Ret% x Hct-patient/45.
Reticulocytes are counted such as young (group I, II) or mature (group III, IV) cells, according to the amount and distribution of stained reticulum. Based on the classification of Heilmeyer and Westhauser (Heilmeyer L. and Westhauser R.: Studies on the ripening of surviving reticulocytes in vitro and their significance in the evaluation of daily hemoglobin production in vivo. Zitschr. Klin. Med. 121: 361-379, 1932) and of Seip (Seip M.: Reticulocyte Studies. Acta Med. Scandinav. Suppl. 282: 9-164, 1953), group I reticulocytes have a dense coherent reticulum filling the greater part of the cell (0-4%). Group II reticulocytes have an extended reticulum, which is looser than that in group I because of occasional breaks in the network (6-22%). Group III reticulocytes have some small bits of reticulum (6- 18%), and group IV cells have only a few blue granules and are essentially erythrocytes (normal value : 78-92%).
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