RNA extraction from fresh and frozen blood
Whole blood contains nucleated leukocytes, so it is easy to extract RNA from blood, but red blood cells are rich in ribonuclease, so it is difficult to isolate RNA from blood. Therefore, it is easier to extract RNA from blood if nucleated leukocytes and red blood cells are separated first. The following methods are acid-phenol-guanidine and guanidine-thiocyanate. Guanidine - Guanidine thiocyanate lyses the cells and effectively inactivates ribonuclease.
Operation method
RNA extraction from fresh and frozen blood
Materials and Instruments
Phosphate buffer Lymphocyte isolate RNAzol B ( Biogencsis UK) Chloroform Isopropanol Ethanol Sodium Chloride DFPC-treated water Centrifuge tubes 1.5X Solvent D: 6 mol L Guanidine thiocyanate Sodium Citrate Sodium Dodecyl Sarcosinate Sodium 2-mercaptoethanol Phenol Sodium Acetate Move I. Materials and equipment Caveat 1) RNAzolB contains guanidinium thiocyanate, which is an irritant; therefore, it is recommended that guanidinium thiocyanate be handled in a fume hood.2) A suitable anticoagulant is 3.8% sodium citrate diluted 1:10 in human whole blood. Heparin may interfere with reverse transcription and PCR.3) The intermediate phase may be so diffuse that the aqueous phase is not visible. If this is the case, the specimen can be left until the intermediate phase is deposited (overnight if necessary). For more product details, please visit Aladdin Scientific website.
Centrifuge Tube
1. RNA extraction from fresh blood
1) Phosphate buffer solution (PBS)
2) Lymphocyte isolation solution
3)RNAzolB (Biogencsis,UK)
4)Chloroform
5)Isopropyl alcohol
7)ethanol
8)5mol/L sodium chloride
9)DFPC treated water
10) 10 ml centrifuge tube
2. RNA extraction from frozen blood
1)1.5X solution D:6mol/L guanidine thiocyanate, 37.5 mmol/L sodium citrate pH7.0,0.75% sodium dodecyl sarcosinate H0.15mol/L 2-mercaptoethanol
2) Phenol: chloroform (5:I, pH about 4.0)
3) 4,2mol/L sodium acetate pH4-0.
4) Isopropyl alcohol.
5) Ethanol.
6) 30 ml centrifuge tube
II Methods of Operation
1. RNA extraction from fresh blood
1) In a 20 ml sterile tube. Dilute anticoagulated whole blood (2:l) 10 mL with phosphate buffer.
2) Carefully add 10 ml of diluted blood to a centrifuge tube containing 3 ml] of lymphocyte isolate. Ensure that there is no visible interface between the blood and the isolate. There should be little or no mixing between the two.
3) Centrifuge at 400 g for 30-40 min or 800 g for 15 min at room temperature.
4) After centrifugation, a clear solution is obtained, with aggregated red blood cells sinking to the bottom of the tube and monocytes including lymphocytes forming a distinct cloud-like band at the interface between the upper plasma phase and the lower lymphocyte isolate. The monocyte layer was transferred to another centrifuge tube using a pipette tip.
5) Add 1XPBS until well mixed and centrifuge as in step 3.
6) Remove the upper layer of solution. In this state, the lymphocytes can be stored at -20°C for several days.
7) Add 0.RMAzolB to lyse the cells, repeatedly blowing with a pipette tip to sufficiently lyse the cells.
8) Transfer to another sterile Eppendorf tube, add 50u1 chloroform, shake vigorously for 15s, and incubate for 5 min at 4℃ or on ice.
9) Centrifuge at 12000 g for 15 min.
10) Transfer the upper aqueous phase to a new Eppendorf tube, be careful to avoid mixing with the intermediate phase, which contains DNA and protein. Add about 400ul of isopropanol of Citrus aurantium et al. and incubate overnight at 4℃ or 20℃.
11) Centrifuge at 12,000 g for 15 mim, and the RNA precipitate will be visible at the bottom of the tube.
12) Remove the supernatant, add 800ul of 75% ethanol to wash the RMA precipitate once, and centrifuge at 7500 g for 8 min.
13) Add 200ul of 0.2mol/L NaCl to dissolve the RNA again, precipitate the RNA with 400ul of 100% ethanol and place at 20℃ for lh.
14) Centrifuge at 12000 g for 15 mm, and wash the RNA precipitate with ethanol as above.
15) Open tube L1 and dry at room temperature for 15 min.
16) Add 50~100ul of DEPC-treated water to dissolve the RNA, to increase the solubility, dissolve the RNA at 52~60℃ for 5~I5 min.
17) Quantitative analysis of RNA
2. R1NA extraction from frozen blood
1) Heat 2 ml (or 5 ml) of frozen whole blood on a water bath.
2) Thaw the specimen well and transfer it to a sterile 30 ml centrifuge tube containing 3 ml (or 5 ml) of 1.5XD solution and mix well. Add 6 ml (or 1Oml) of phenol:chloroform (5:1) and 0.5 ml (or 1 ml) of 4.2 mol/L sodium acetate, pH 4.0, and mix well, preferably with a sealing film.
3) Incubate on ice for 20mim.
4) Centrifuge at 5500 g, 4°C for 30 min.
5) Transfer the supernatant to another centrifuge tube
6) Add an equal volume of isonairesinol, and leave overnight at 20℃.
7) Centrifuge at 5500 g for 30 min.
8) Discard the supernatant and invert the tube for 2~3 min.
9) Add 300ul of 1.5X solution D to dissolve the precipitate. Add 400ul of isopropyl alcohol if necessary and place at -20°C for lh.
10) Centrifuge at 11000 g for lOmin at room temperature.
11) Discard supernatant, add 750ul of 75% ethanol to wash the precipitate and centrifuge at 11000 g for 2 min.
12) Discard the supernatant and add 50ul of DEPC treated water to dissolve the RNA.
13) Quantitative analysis of RNA