Hidradenitis elegans nematode RNA preparation
Hidradenitis elegans nematode RNA preparation
A modification of the standard method for RNA preparation from mammalian tissues and Drosophila that focuses on rapid lysis of Juniperus communis and homogeneous purification of RNA.
Operation method
Preparation of RNA from the nematode Hidradenitis elegans (Hidradenitis elegans)
Materials and Instruments
DEPC treated water. l.0mol LTris solution (pH 7.5) TE buffer TE buffer with 0.1% SDS 20% sodium dodecylbenzene sulfonate homogenization buffer Cesium chloride solution 3mol L sodium acetate (pH 5.2) anhydrous ethanol Ethanol Move I. Materials and equipment Caveat The yield of RNA from nematode RNA extracted by this method is 0.8 to l.lmg of pure total RNA per ml of compacted worm body. The product may contain some contamination with K.coliRNA contained in the starting material, and the percentage of E.coliRNA in the product can be determined by gel electrophoresis, hybridization, etc. The position of nematode rRNA is 3.5 to 1.7 kb, so that it can be distinguished from E.coliRNA, which is electrophoretically located at 3.0 to 1.5 kb. The position of nematode rRNA is 3.5kb~1.7kb, which can be distinguished from E.coliRNA with electrophoretic position of 3.Okb~1.5kb. If the nematode is cultured in liquid, the content of E.RNA in the final product will be 0~40%. For more product details, please visit Aladdin Scientific website.
Brinkmann homogenizer BeckmannL8-80 centrifuge
Distilled and sterilized deionized water is required for all buffer preparations.
1) DEPC treated water.
2) l.0mol/L Tris solution (PH 7.5), prepared with DEPC-treated and autoclaved water, but not Tris solution.
3) TE buffer.
4) TE buffer containing 0.1% SDS.
5) 20% sodium dodecylbenzene sulfonate. Add DEPC to a final concentration of 0.1%, mix well with shaking, and autoclave overnight at 37°C for 20 min to destroy DEPC.
6) Homogenization buffer: 4.Omol/L guanidine isothiocyanate, 0.1 mol/L Tris (pH 7.5). After preparation, remove particulate matter with a sterilized filter. Before use, add β-mercaptoethanol to a final concentration of 1%.
7) Cesium chloride solution: composed of 5.7mol/LCsCl and 0.Olmol/LEDTA (pH8.0). To prepare, dissolve 96 g of CsCl and 2 ml of 0.5mol/LEDTAC (pH8.0) in about 70 mlddH20, volume to 100 ml and remove particulate matter using a sterilized filter, transfer the solution to a bottle and carefully mark the position of the concave liquid level. DEPC was added to a final concentration of 0.1%, mixed with shaking, and incubated at 37°C overnight. Finally, autoclave for 20 min to destroy the DEPC. After autoclaving, the volume of the solution will be reduced by evaporation of the DEPC. Add DEPC-treated water to the marked concavity to make up the solution and mix well.
8) 3mol/L sodium acetate (pH 5.2). Add DEPC to a final concentration of 0.1%, mix with shaking, leave overnight at 37°C, and autoclave for 20 min to destroy DEPC.
9) Anhydrous ethanol.
10) 70% ethanol without RNase contamination.
11) Brinkmann homogenizer
12) Beckmann L8-80 centrifuge.
II Methods of operation
1) Add 1 to 5 ml of worm suspension (50% of the worms dissolved in 0.lmol/l NaCl) to a 50 ml centrifuge tube, if more than 5 ml of worm suspension is used, the suspension will overflow during homogenization. Preferably, freshly prepared nematodes or freshly frozen and stored at -80°C should be used.
2) Use an electric tissue homogenizer to lyse the worms. Homogenize rapidly in denaturing solution that inactivates RNase to maximize degradation of RNA.
3) Add 5 times the volume of homogenization buffer (β-mercaptoethanol should be added to the homogenization buffer) to the fresh nematode suspension and start homogenization immediately. For frozen nematodes, add the buffer directly to the frozen nematodes. Start homogenization immediately. Start homogenization by turning the homogenizer to a lower speed and inserting the cobalt tip into the frozen nematode. This will allow them to be broken up and suspended from the bottom of the tube. As soon as the bulk of the frozen material has been dispersed, the homogenizer should be turned to maximum speed and homogenized for 2 min.
4) Add sodium dodecylbenzene sulfonate solution to a final concentration of 0.5% and mix well.
5) Place the mixture in a 28 ml centrifuge tube and centrifuge at 30OOOr/min for 20 min at 20°C to remove the precipitate. The supernatant is then transferred to a clean 50 ml centrifuge tube. The supernatant should be yellow-brown in color.
6) A density gradient of cesium chloride should be prepared for every 6 ml of homogenized product. Preferably use RNase-free 16 mmX102 mm polyallomar centrifuge tubes that can be used in a SW28.1 turntable rather than ultraclear tubes, which are prone to rupture in centrifugation. 11.5 ml of cesium chloride solution should be added to each centrifuge tube with a plastic pipette and the tube walls should be tapped lightly to remove air bubbles that may be present in the solution. The plastic pipette was used to add 11.5 ml of cesium chloride solution to each tube and the tubes were tapped to remove any air bubbles in the solution that might have gotten on the walls.
7) Draw the nematode homogenate product into a 5 ml syringe fitted with a standard 23-gauge needle and spread it gently along the wall of the centrifugal curfew over the surface of the cesium chloride density gradient to a point 1 to 2 mm from tube U1 . Approximately 8 ml of homogenate can be added to each tube, if the homogenate does not fill the tube. If the tube is not filled with homogenization product, make up with homogenization buffer containing 0.5% sodium dodecylbenzene sulfonate. Mark the contact surface between the cesium chloride solution and the homogenate with a marker.
8) Carefully place the centrifuge tube into the SW28.1 centrifuge bucket, tighten the lid, and load the bucket onto the rotary head of the ultra-high speed centrifuge (care should be taken not to disturb the density gradient of cesium chloride during this process). When centrifuging at 27000r/min at 20℃, do not use fast acceleration and rapid deceleration, so that the density gradient of cesium chloride is not disrupted as much as possible. In practice, the centrifugation time can be lengthened or shortened depending on the circumstances.
9) After centrifugation, carefully transfer the centrifuge tube to a rack (be careful not to destroy the gradient). The brown protein should remain above the markings made by the marker and there should be a white double ring band in this brown material. There should be another white band in the clarified portion of the cesium chloride density gradient, which is nematode DNA. the RNA should be a clear substance loosely adsorbed to the bottom of the tube (the RNA is only available after almost all of the liquid has been removed), and the clumps of RNA should look like thin, shiny, low-melting-point agarose in a volume of about 30 ul.
10) Removing the liquid requires a great deal of patience, both to avoid contamination of the RNA by the upper layers of the cesium chloride density gradient and to prevent the loss of the very difficult to read RNA clumps. Use a 10 ml plastic pipette and a rubber bulb to carefully aspirate the upper layer up to the marker. The pipette should be pipetted with the mouth of the pipette close to the liquid surface so that liquid and air are drawn up together to ensure that the least dense and most contaminating substances at the top of the gradient are removed first, so that they do not fall out with the lower layer as it is drawn up. Change to a new pipette and aspirate more liquid in the same way up to the lower side of the circular DNA strip. Change to another new pipette and aspirate most of the remaining liquid. Leave about 2 ml of liquid just over the bottom of the bow of the centrifuge tube, and the remaining liquid should be pipetted out with extreme caution to prevent loss of RNA.
11) Using forceps, take a clean surgical blade and cauterize it on a Bunsen burner until it turns red, then use it to cut (actually melt) the tube. The cutting point should be just above the top of the liquid level of the remaining liquid. When separating the top from the bottom of the tube, a small section of the tube should be left where they meet, so that the bottom of the tube can be easily picked up using the top as a handle. The purpose of removing the 0 from the top of the tube is twofold: first, to separate the contaminated upper wall from the uncontaminated bottom, and second, to help visualize the RNA clumps at the bottom.
12) Remove the remaining liquid very carefully with a 20-ul pipette that is free of RNase contamination. At this point, the clear, gel-like RNA clumps may be floating or in sheets. During the experiment, tilting the bottom of the tube in all directions helps to separate the liquid from the RNA pellets so that the liquid can be safely pipetted out. Sometimes it is almost impossible to aspirate the last ~30ul of liquid without aspirating any RNA at all. In this case, leave the last bit of liquid T and add about twice the volume of room temperature anhydrous ethanol (without RNase). This will cause the remaining cesium to form a white precipitate with the RNA and will better coat the bottom of the centrifuge tube. The next step can be continued (filling the tube with 70% ethanol) All residual cesium chloride will eventually be removed in the ethanol precipitation in the next step.
13) Fill the bottom of the centrifuge tube with 70% ethanol at room temperature. This step will leach out the remaining Cesium Chloride, and the RNA will turn white after two minutes and adhere more tightly to the tube wall.
14) Pour off or aspirate the remaining liquid and dry the RNA precipitate for a short time. When the RNA starts to become clear, add 175ul of TE solution containing 0.1% SDS to each tube and blow up and down repeatedly with a pipette to suspend and dissolve the RNA. this process should continue for 1 mim. after that, transfer the RNA to a new RNase-free centrifuge tube with a screw cap and add another 25ul of TE solution containing 0.1% SDS to the original tube and blow several times. TE solution containing 0.1% SDS was added to the original centrifuge tube and blown several times. Finally, the two parts of the RNA solution are combined. If the RNA is still not completely dissolved (sometimes some small pieces remain), freeze it in liquid nitrogen and thaw it in a 50°C water bath once or twice to completely dissolve the RNA.
15) For 200ul of RNA solution, add 150ul of TE, 30ul of 3nol/L sodium acetate (pH5.2) and 900UL of ethanol to precipitate the RNA. Centrifuge the solution at -20℃ for 3Omin. 4℃ for lOmin. Discard the supernatant, rinse the precipitate with 70% ethanol, dry it, and then use a small amount of water to dissolve the RNA (usually about 80ul, depending on the amount of RNA). 80ul, depending on the amount of RNA).
16) Store the RNA at -80℃.