Preparation of blood samples for gene chip analysis
Preparation of blood samples for gene chip analysis can be used for (1) gene expression analysis and (2) analysis of gene expression in blood leukocytes.
Operation method
Preparation of blood samples for gene chip analysis
Principle
The prototype of genechip (also known as DNA chip, biochip) was proposed in the mid-1980s. The sequencing principle of genechip is hybridization sequencing method, i.e., the method of nucleic acid sequence determination by hybridization with a set of nucleic acid probes of known sequence, which are fixed on the surface of a piece of substrate with a probe of target nucleotide of known sequence. When the nucleic acid sequence TATGCAATCTAG, which is fluorescently labeled in the solution, produces a complementary match with the nucleic acid probe at the corresponding position on the gene chip, a set of probe sequences whose sequences are completely complementary is obtained by determining the position of the probe with the strongest fluorescence intensity. Accordingly, the sequence of the target nucleic acid can be recombined.
Materials and Instruments
Blood Samples Move 1. 2 mL of anticoagulated blood (heparin or EDTA salt anticoagulation is acceptable) will be centrifuged at 3,500 rpm for 10 minutes. The blood will be divided into 2 layers, the upper layer being yellowish plasma, which is about 60% of the blood. 2. Use a bus dropper or pipette to suck out the upper plasma layer, leaving about 1 ml of blood cell layer. Add 1:1 volume, i.e., about 1 ml of sterilized saline to the blood cell layer and mix well. 3. The following is the process of separating leukocytes after combining 12 ml of saline with the blood cell mixture (equivalent to 12 ml of whole blood) into one tube. Add 24 mL of lymphocyte isolate (twice the volume of whole blood) to a sterilized 50 mL centrifuge tube. Blood cells mixed with saline are slowly added to the surface of the lymphocyte separation fluid, taking care not to mix the blood cell layer with the lymphocyte separation fluid layer, as shown in Figure 3; the fluid is then centrifuged for 20 minutes at 1500 rpm (centrifuges require a horizontal rotor head). It can be seen that the liquid is divided into three layers, the upper layer is yellowish plasma, the middle layer is milky white, and the bottom layer is red blood cells. At the junction of the middle layer and the upper layer, there is a white membrane, i.e., the leukocyte layer, and at the same time, there is a thicker milky white layer of liquid under the obvious leukocyte layer, which also contains leukocytes, but the amount is relatively small, if there is enough blood, you can consider not to have this layer (if the leukocyte layer is not very obvious and the milky white liquid layer is too thick, you can centrifuge it at 2,500 rpm and continue to centrifugate for 10 minutes in order to strengthen the effect of separation of the leukocytes). 4. Collect the leukocyte layer from the interface with a bus dropper or pipette, place in a sterilized 1.5 mL EP centrifuge tube, centrifuge at 10,000 rpm for 5 minutes, and aspirate the supernatant. The supernatant should be aspirated. A white precipitate should be seen, sometimes with red blood cells mixed with the white precipitate (a few red blood cells are not harmful). 5. Remove the supernatant and add 1ml of Trizol reagent to the cellular precipitate, pump well with a pipette and mix well until a clear, non-sticky liquid is formed (indicating that the cells have been sufficiently lysed and the genomic DNA has been sufficiently interrupted). 6. Total RNA can be extracted according to the standard operation of Trizol reagent, or this Trizol reagent can be stored in biological ice (below 15℃ can be) and mailed to us, we will complete the subsequent extraction process. Generally, 10-15ug of total RNA can be obtained per 5 ml of whole blood. Caveat 1. Lymphocyte isolate is generally required to be stored in 4℃. Lymphocyte isolation solution should not be applied immediately after it is taken out of the refrigerator to avoid cold stimulation of the cells, which may cause changes in gene expression. Wait until the solution temperature rises to room temperature, then mix well and use. 2. The temperature should be 18-28℃ during the whole isolation process; too high and too low temperature will affect the quality of isolation. Common Problems 1. In some methods, the erythrocytes are separated by centrifugation after adding lymphocyte isolation solution directly to whole blood; the leukocyte precipitate obtained in this way carries more plasma proteins, which affects the extraction of RNA, therefore, we suggest using the above method, discarding the plasma first and then adding lymphocyte isolation solution. 2. In some methods, the isolated leukocytes are preserved in RNAlater for transportation; since the isolated leukocytes are often contaminated with erythrocytes, the erythrocytes will affect the preservation effect of the RNAlater; in addition, some of the leukocytes preserved in the RNAlater will be lost in the RNAlater when it is re-cycled to extract RNA. Therefore, if you have Trizol reagent in your lab, we recommend lysing the leukocytes directly with Trizol reagent. RNA in Trizol reagent is relatively stable, and can be preserved at -20℃ for one week and at -70℃ for one month. For more product details, please visit Aladdin Scientific website.
Lymphocyte isolate Sterilized saline Trizol
Centrifuge