Experimental procedure 1. All containers are sterilized twice at high temperature, DEPC is sterilized at high temperature, all reagents are prepared with DEPC and sterilized at high temperature. 2. Add to a sterile 2mL tube: 5mol / L guanidine isothiocyanate 0.7mL, phenol 0.4mL, NaAc (pH4.0) 0.1mL. 3. Weigh 0.2g of wheat yellow seedlings, quickly grind them into powder in liquid nitrogen, transfer them to the prepared centrifuge tube, and shake vigorously. 4. Mix well, ice bath for 30min. 5. 4 ℃, 12000rpm, 10min. 6. Add supernatant to another centrifuge tube and add 0.4mL chloroform, shake vigorously. Place in an ice bath for 5 minutes. 7. 4 ℃, 12000rpm, 10min. 8. Discard the organic phase (lower layer), add 0.4mL chloroform and 0.4mL phenol, and leave at room temperature for 5min. 9. 4 ℃, 12000rpm, 10min, discard the organic phase and add an equal volume of isopropanol. Place at -20 ℃ for 1h. 10. 4 ℃, 12000rpm, 10min, the precipitate was washed twice with 70% ethanol. 11. Slightly dry at room temperature. 12. Precipitate and add 20μL DEPC water to dissolve (store at -20 ℃) 13. RNA electrophoresis: 1% agarose gel 20mL, 8μL sample, 1μL sample buffer, 1μLSYBR, 100V constant voltage electrophoresis, measuring OD260 and OD260 / OD280. Note 1. The purpose of step 1 is to remove the influence of RNase (exogenous). High temperature sterilization twice can destroy the renaturation process of RNase. Although RNase is very easy to renature, two consecutive high temperatures will disrupt its renaturation process, thereby inactivating RNase. DEPC is a non-competitive inhibitor of RNase. DEPC can bind to the N on the histidine imido ring of the active center of RNase, thereby inactivating the RNase. Because DEPC can bind to the N of RNA, thereby modifying the RNA, the DEPC must be finally removed. During high temperature sterilization, DEPC evaporates due to high temperature decomposition. UV can also modify RNA and should be avoided during experiments. DEPC is called DEPC water after sterilization, it is free of DEPC. Change gloves frequently during the experiment, if necessary, in the ultra-clean workbench. 2. The denaturant must be prepared in advance. Guanidine isothiocyanate can denature RNase and can also break cells. This test can not be used to break cells enzymatically. Because suitable conditions for proteinase K can also make RNase active. NaAc acid can make DNA in the organic phase (phenol phase), and under alkaline conditions, DNA and RNA are in the aqueous phase, can not be separated. Phenol is used to extract DNA and protein. 3. Low temperature prevents endogenous RNase from degrading RNA. Vigorous shaking is to loosen all the cells and immerse them in denaturant. Low temperature cells will form a clump in the relatively high temperature liquid, so that the internal RNA will not have the opportunity to hydrolyze RNA. The yellowed wheat seedlings do not have photosynthesis, so they contain less sugar and are easy to extract. 4. The purpose of step 6 is to remove lipids. 5. The purpose of step 8 is to remove lipids and proteins. 6. The purpose of step 9 is to remove sugar and dehydrate. Because the system is high in salt (2mol / L NaAc), RNA can be precipitated. 7. Step 10 Note that because the amount presented is small and may not be visible, the centrifugation should have a direction mark. 8. RNA is very difficult to dissolve, so it can only be dried slightly. If it does not dissolve because there is too much sugar left. 9. DEPC water should be free of RNase.
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