Protocol for RNA Extraction from Peripheral Blood Lymphocytes
GUIDELINE
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. Peripheral blood lymphocytes (PBL) are mature lymphocytes that circulate in the blood, rather than localizing to organs (such as the spleen or lymph nodes). They comprise T cells, NK cells, and B cells.
METHODS
- Fresh human blood was collected and leukocytes were immediately separated with Ficoll.
- PBLs were washed 3 times with ice-cold PBS.
- Collect peripheral blood lymphocytes in a 50 ml plastic centrifuge tube and add 7 ml of lysis buffer (lyses up to 5×108 PBLs), then shake vigorously with a vortex to lyse the cells.
- Add 7 volumes (49 ml) of 4 mol/L lithium chloride and incubate at 4°C for 15-20 hours (overnight).
- The suspension was transferred to a 30 ml Corex tube and centrifuged for 2 h at 6500 r/min at 4°C in a boom-type rotor.
- The supernatant was discarded and the mouth of the tube was wiped with Kimwipe. Resuspend with 3 mol/L lithium chloride (approximately 15 ml) and collect the precipitate. Centrifuge the precipitate resuspension at 6500 r/min for 1 hour.
- Discard the supernatant and lyse the precipitate with 2ml RNA lysis solution. Freeze the suspension thoroughly at -20°C.
- Resolubilize the suspension, and vortex for 20 seconds every 10 minutes for 45 minutes.
- Extract once with an equal volume of phenol and once with an equal volume of chloroform.
- Add 1/10 volume of 3 mol/L sodium acetate, pH 4.8, and 2 volumes of one 20°C ethanol. Mix the solution thoroughly and incubate at -20°C overnight.
- Centrifuge the RNA in a boom head at 12,000 r/min for 30 min. Resuspend the precipitate with 0.2 ml DEPC-treated water. Transfer the dissolved DNA to a 1.5 ml microcentrifuge tube and reprecipitate by adding 1/10 volume of 3 mol/L sodium acetate, pH 4.8, and 2 volumes of -20°C ethanol. and preserve the RNA as an ethanol precipitate until ready for use.
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NOTES
- Centrifuge tubes, Tip tips, pipette stems and electrophoresis baths involved in the experiment, and the lab bench top should be thoroughly disposed of.
- The reagents or solutions involved in the experiment, especially water, must be ensured to be RNase-free.
- Control the starting amount of the sample.
