Extraction method of human peripheral blood lymphocyte RNA

Extraction method of human peripheral blood lymphocyte RNA

First, collect blood samples and store at room temperature. Blood was collected using a disposable anticoagulated vacuum pumping blood vessel. Generally, EDTA or heparin is used for anticoagulation; the blood volume is generally 1 ml ; in general, 1 ml of peripheral blood contains about 1-2*10^6 mononuclear cells ( PBMC , ie, lymphocyte nuclear monocytes).

Second, extract lymphocytes. Take 1.5ml   Sterile enzyme-free centrifuge tube, tube 1 , first add 500ul lymphocyte separation solution; another 1.5ml   In a sterile, enzyme-free centrifuge tube, a 500 ul blood sample vacuum tube and 500 ul PBS (phosphate buffer), 1 : 1 mixed mixture, 1 ml total. Then slowly add the mixed blood sample PBS mixture to the tube 1 with lymphocyte separation solution, pay attention to be slow, so that the blood sample is located in the upper layer of the extract as much as possible, and can not force the blood cells to spread to the lower layer too much, there is no way to centrifuge and stratify. . (This step should be done as quickly as possible, because the red cells will settle under gravity, mix into the extract, affect the subsequent centrifugation) and then centrifuge. 1500 rpm, 15 min, 20 degrees Celsius .

Description:

1. It is best not to exceed 4 hours from the time of collecting the blood sample to the start of the experiment. The room temperature condition is maintained throughout the experiment, generally 20 degrees Celsius is moderate, because the optimal temperature of the cells is 37 degrees Celsius, but the temperature can reduce the metabolism of the cells, and there are some contradictions, so the intermediate temperature is ideal.

2 , note on lymphocyte separation solution: should be stored at 4 ° C after unsealing to avoid microbial contamination; after the separation solution is taken out of the refrigerator, it can not be used immediately, need to wait until the temperature of the solution rises to room temperature, shake it and use it; During the process, the temperature should be controlled at 18-28 ° C and in a sterile environment to avoid microbial contamination, otherwise the quality of the separation will be affected; it will be stored at 18-25 ° C in the dark before unsealing, and stored at 4 ° C after unsealing. For vacuum packaging, placed at 10 ° C before unopening   The following white crystals are prone to occur, which affects the separation effect. (The storage temperatures of various lymphocyte separation solutions are not exactly the same)
Lymphocyte separation solution is a commonly used reagent for separation and purification of cells according to the difference in cell density and gravity acceleration by centrifugation. It is a sterile aqueous solution with opalescence or microemulsion. The main components are dextran and pan. Glucosamine. It is suitable for the isolation and purification of human lymphocytes and most mammalian cells, and can remove red blood cells and dead cell debris. The obtained PBMC can be further used for primary culture or flow cytometry. The most commonly used cell separations are Ficoll and Percoll .

3 , the number of revolutions is 1500 rpm , the time range is 15 minutes, and the temperature is room temperature 20 degrees Celsius. Two-step centrifugation is recommended: first centrifuge at 1500 rpm/min for 15 min . If the tunica layer is enough to complete, if the cells are not enough, the patient's lymphocyte specific gravity is low, and then centrifuge again at 1500 rpm / min for 15 min .

Third, after centrifugation, divided into four layers. Lymphocytes are located on the second layer (from top to bottom) and are a thin layer of white. Use a pipette ( 200ul file) to absorb the lymphocytes in the separation layer. The technique is relatively strong. It requires more practice to compare the lymphocytes to the more complete, and about 400ul of liquid can be obtained. The aspirated lymphocyte solution is placed in another sterile centrifuge tube. Add 1 ml of PBS buffer to the centrifuge tube, 1500 rpm , 15 min , 20 ° C. Pour off the supernatant liquid after centrifugation, repeat the centrifugation step until the liquid is not red (ie, the number of red blood cells is very small). The lymphocytes are located at a small amount of white matter at the bottom of the centrifuge tube, usually mixed with a small amount of red blood cells, and avoided as much as possible.

Description:

1. The conditions for centrifugation after washing are usually sufficient at 1500 rpm. If it is too high, it is easy to kill the cells. The time is 15 minutes and the temperature is still 20 degrees Celsius.

2 , after centrifugation, see a small amount of red blood cells mixed in the lymphocytes, the problem is not big in this experiment.

3 , the choice of washing solution is also balanced salt solution, the main purpose is to remove plasma proteins and platelets in lymphocytes, red blood cells are difficult to remove. Generally, the washing solution PBS can be used in 3 ml .

Fourth, use Trizol reagent   Lymphocytes are lysed and RNA is extracted. Add 1ml of Trizol reagent to the centrifuge tube to lyse other macromolecules other than RNA , cell membranes, organelles, etc. This thing is carcinogenic and teratogenic. Wear a mask and handle it carefully. Blot the lymphocyte mass at the bottom of the tube with a liquid gun and break it up. Usually at least 30-60 times, the main purpose is to break it up and let Trizol reagent fully lyse lymphocytes. Transfer the liquid after the blow to the cryotube, mark it, soak it in liquid nitrogen (this step is to freeze the mRNA quickly), and after one to two hours, transfer to the liquid.   -80 degrees Celsius in the refrigerator.

Five, delivery

Description:

1. TRIZOL reagent is a reagent for extracting total RNA directly from cells or tissues. It maintains RNA integrity when breaking and lysing cells.

2. In TRIZOL , RNA is isolated from RNase contamination. Subsequent manipulation of the sample may require the use of RNase -free non-disposable glassware or plastic vessels. The glassware can be baked in an oven at 150 ° C for 4 hours. Plastic utensils can be at 0.5 M   Soak in NaOH for 10 minutes, rinse thoroughly with water and autoclave for use. A clean disposable polypropylene tube is recommended when the TRIZOL dosage is less than 2-ml .

3. Finally, use the frozen tube with sterile enzyme-free tube for the cryotube containing the lysed cells.