Original Title: RNA Extraction and Preparation Methods for Common Medical Samples As a kind of very important biological macromolecule, nucleic acid is the master in the biological world. It plays an important role in all life activities such as reproduction, development, maintenance, aging and death of organisms. If there is a slight change in its structure, such as an error, deletion or addition of a base, It may lead to mutation, defect or disease, so nucleic acid has become a hot research topic in biochemistry, genetics and other related life disciplines, and its wide coverage and rapid progress are beyond the reach of other disciplines. The first prerequisite for nucleic acid research is to extract nucleic acid from many complex biological macromolecules and purify it to a certain extent so as to carry out corresponding scientific research and practical application. Nucleic acid, coming to the "tube", is divided into six phases. Through this series, we share with you the knowledge of sample preparation, extraction methods and detection of nucleic acid extraction, in order to be helpful to your scientific research. 1 Whole blood sample 01 Selection of blood collection tube One of the major problems with blood collection for testing is the instability of intracellular RNA, which degrades rapidly within hours of blood collection. In addition, RNA from some species is increased in vitro by genetic induction after blood collection. Both in vitro RNA degradation and gene induction can lead to an underestimation or overestimation of the number of transcripts of relevant genes in vivo. BD's PAXgene blood RNA tubes contain additives that stabilize gene transcription in vivo, reduce RNA degradation in vitro, and minimize the level of gene induction. They are suitable for human and primate whole blood sample preparation. For whole blood samples, we only recommend the use of this tube. BD PAXgene Blood RNA Tube (Item No.762165) 02 Collect blood Please read the product instructions carefully before using the PAXgene blood RNA tube for correct operation. If the PAXgene Blood RNA tube is the only blood drawing tube, the blood should be drawn into a "waste tube" prior to being drawn into the PAXgene Blood RNA tube so that the phlebotomizer used in the blood drawing process can be prefilled with blood; otherwise,rotovap distillation, the PAXgen Blood RNA tube should be the last tube in the blood drawing procedure. Make sure that the blood has stopped flowing into the tube before removing the tube from the needle holder (the negative pressure in the PAXgene Blood RNA tube is designed to draw 2.5ml of blood into the tube). Immediately after blood collection, the PAXgene blood RNA tube should be gently inverted 8-10 times to ensure that the protective agent and blood in the tube are well mixed. Expand the full text 03 Preservation and transportation PAXgene blood RNA tube are allowed to stand vertically at room temperature (18-25. degree. C.) for 2-24 hour, after which they may be stored at -20. degree. C. or lower; if that tube are to be stored at a temperature lower than -20. degrees. C., the tubes may be frozen at -20. degrees. C. for 24 hours, and then transferred to -70. degree. C or -80. deg. C for at least 50 months. Bulk dry ice transport. 2 Serum Plasma Samples Because the abundance of RNA in cells is much higher than that of free RNA in blood, even a very small amount of cells will have a great impact on the analysis of free RNA in blood. Therefore, removing the contamination of cells and cell debris is the key to the preparation of serum plasma samples. 01 Selection of blood collection tube For serum samples, it is enough to use common serum vacuum blood collection tubes; for plasma samples,wiped film evaporator, it is recommended to use EDTA anticoagulant tubes, and heparin anticoagulant tubes are prohibited. 02 Separation and storage of plasma samples Collect blood samples with EDTA anticoagulant tube (the collected blood samples can be stored at room temperature or 4 ℃ for a short time, but not more than 1 H). 1900 G (or 3000 rpm), 4 ℃, centrifugation for 10 min. Carefully transfer the upper plasma layer (yellow) into a new 1.5 ml centrifuge tube without the tip of the gun touching the middle layer (white blood cell and platelet layers). In general, 4 to 5 ml of plasma can be separated from 10 ml of blood. The separated plasma was centrifuged at 16000 G, 4 ℃ for 10 min. Do not touch the impurities on the bottom side of the tube with the gun head. Carefully transfer the supernatant to a new 1.5ml centrifuge tube and store it at -80 ℃. Bulk dry ice transport. Plasma samples were isolated 03 Separation and preservation of serum samples Blood samples were collected with blood collection tubes containing coagulation activators or ordinary vacuum blood collection tubes, and the blood was allowed to coagulate at room temperature for 10-60 min. 1900 G (or 3000 rpm), 4 ℃, centrifugation for 10 min. Carefully transfer the upper serum (yellow) to a new 1.5 ml centrifuge tube without the tip of the gun touching the middle layer (white blood cell and platelet layers). In general, 3-5ml of serum can be separated from 10ml of blood. The separated serum was centrifuged for 10 min at 4 ℃ with a 16000 of G. Do not touch the impurities on the bottom side of the tube with the gun head. Carefully transfer the supernatant to a new 1.5ml centrifuge tube and store it at -80 ℃. Bulk dry ice transport. Serum sample separation 3 cell supernatant Cell culture media or other biological fluids are collected using a suitable container. 3000 G, 4 ℃, 15 min. Carefully transfer the supernatant to a new sterile centrifuge tube. The supernatant was centrifuged at 4 ℃ for 10 min with a 16000 of G. Do not touch the impurities on the bottom side of the tube with the gun head. Carefully transfer the supernatant to a new sterile centrifuge tube and store it at -80 ℃. Bulk dry ice transport. Cell supernatant separation 4 Exosomes For the teacher's own enriched exosomes, it is necessary to dissolve the exosomes in not more than 100ul RNase-free PBS and mix them well to facilitate subsequent extraction. The exosomes sample solution was stored at -80 ° C. Bulk dry ice transport. 5 Tissue samples For oral tissue samples used for RNA extraction, if the sample volume is small, it is recommended to give priority to the sample delivery method of protective solution (RNAlater, cbd centrifugal extractor ,wiped film distillation, etc.). Scheme I. Sample delivery of protective solution (RNAlater, etc.) The fresh tissue was cut into samples with length, width and height ≤ 0.5 cm with a scalpel, and small organs such as mouse liver, kidney and spleen could be preserved intact in RNA later. Immerse the tissue sample in 5-10 times the volume of the RNAlater to completely submerge the tissue. Samples were incubated overnight at 4 ° C (to allow complete tissue penetration by RNAlater) and then transferred to -80 ° C for long-term storage. Bulk dry ice transport. Scheme II: Liquid nitrogen quick freezing and sample delivery Fresh tissue was removed, and tissue types not required for the study, such as connective tissue and adipose tissue, were immediately removed. If the tissue volume was large, the tissue should be cut into small pieces (soybean size) with length, width and height ≤ 0.5 cm. The tissue surface was quickly flushed clean of residual blood with precooled PBS solution (RNase free) or saline. The processed tissue samples are mixed evenly and stored in a liquid nitrogen pre-freezing storage tube with a screw cap. Quickly frozen in liquid nitrogen for about 3 H, and then transferred to -80 ℃ for long-term storage. Bulk dry ice transport. Caution: 1. For For the cancer samples of RNA projects, we suggest that the tissues removed by surgery should be quickly put into RNAlater and incubated at 4 ℃ for 24 hours, then the RNA later should be discarded, the tissues in the necrotic area and the paracancerous tissues should be cut off and transferred to the liquid nitrogen pre-freezing storage tube with a screw cap. The liquid nitrogen is quickly frozen, stored at -80 ℃, and transported with dry ice. 2. After the tissue sample leaves the living body It is suggested that liquid nitrogen quick freezing should be carried out within 3 min. The longer the operation time before quick freezing, the greater the possibility of RNA degradation. 3. Tissue samples are not recommended TRIzol sample delivery, if necessary, need to be fully ground in liquid nitrogen and dissolved in an appropriate amount of TRIzol, tissue samples should not be excessive, after 5 minutes of pyrolysis at room temperature, frozen at -80 C, dry ice transportation. 6 cell sample The number of cells required for one RNA extraction reaction is less than or equal to 1 X 10 ^ 7, and the number of cells required for one RNA extraction reaction is preferably between 3 X 10 ^ 6 and 1 X 10 ^ 7. If the sample volume is large, it is recommended to divide the cell sample into different tubes according to the above requirements. 01 Adherent cells Take out the adherent cultured cells from the incubator, observe the cells under the microscope, and confirm that the growth state is good (the normal cell fusion degree is about 80%). The medium was discarded, and 5 mL of PBS (RNase-free, room temperature) was added to the cell culture flask or dish and washed once. Discard the PBS, add a proper amount of TRIzol, and repeatedly suck and beat for several times until the agglomerated cell mass is not visible, so that it can be fully dissolved in the lysis solution to form a clear and non-viscous liquid. The lysed cell solution was transferred to a 1.5 mL screw-cap centrifuge tube (RNase-free), stored at -80 ℃ for a long time, and transported in large volume of dry ice. 02 Suspension cells First of all, make sure that the cells are growing well; centrifuge to get the cell sediment (according to the steps of cell centrifugation in the customer's laboratory, note that the cell centrifugation should be moderate, and do not make the cell centrifugation too solid to cause the lysate can not fully penetrate). Discard the medium, add 1 mL of PBS (RNase free, room temperature), gently suspend the cell pellet, and transfer to a 2 mL screw-top, tip-bottom centrifuge tube (RNase free). Centrifuge (according to the steps of cell centrifugation in the customer's laboratory, note that the cell centrifugation should be moderate, and do not make the cell centrifugation too solid to cause the lysate can not fully penetrate) to obtain cell sediment, discard PBS, add appropriate amount of TRIzol, and repeatedly suck and beat for several times until no agglomerated cell mass can be seen, so that it can be fully dissolved in the lysate. It is stored at -80 ℃ for a long time and transported in large volume of dry ice. Caution: 1. If it is DNA items can be washed with ordinary sterile PBS. If it is RNA items, it must be RNA se-free. 2. Trypsin treatment is not recommended for obtaining cell Pancreatin is protein, and if there is any residue, it will affect subsequent experiments. 3. The number of cells is less than 5 × 105 It is recommended to send the sample directly after quick freezing (without lysis with lysis solution). Due to the small number of cells,50l rotovap, we will arrange micro-extraction. 4. Cell samples are not recommended to be stored in RNAlater tissue RNA protection solution, because of the high density of RNAlater, the cells stored in it are difficult to be collected by centrifugation for extraction. Return to Sohu to see more Responsible Editor:. toptiontech.com