RNA Laboratory Techniques Manual (Molecular Cloning - Laboratory Guide Series)
Operation method
Extraction and purification of RNA from polysaccharide-rich plant tissues
Principle
Guanidine thiocyanate lyses cells and denatures proteins. When used as an extraction buffer, it produces an RNase-free environment □ After tissue extraction, centrifuge the homogenate at medium speed to remove small soluble polymorphs. The supernatant is extracted with phenol:chloroform, with the RNA in the aqueous phase and the DNA and proteins in the phenol phase and at the interface of the two phases. Selective pieces of polysaccharide in the aqueous phase were precipitated with potassium acetate, and the RNA was then selectively purified from residual contaminants with lithium chloride. The effectiveness of this method was demonstrated by the successful extraction of leaf RNA from a variety of plants grown in a high CO2 concentration environment.
Materials and Instruments
0.75mol L sodium citrate N-lauroyl sarcosine Thiocyanoguanidine buffer Extraction buffer Chloroform Isoamyl alcohol 2mol L sodium acetate Acid phenol Isonaiol Ethanol 2mol L potassium acetate 10mol L LiCl TNE buffer TE buffer Liquid nitrogen Move I. Materials and equipment Caveat 1) Initial centrifugation removes most of the insoluble polysaccharides, such as starch granules. The residual tissue forms a dark green precipitate at the bottom of the tube (if leaves were used), and the insoluble polysaccharides form an off-white gelatinous layer on top of the residual tissue.2) When aspirating the supernatant, great care should be taken not to disturb the gelatinous precipitate as the layer is very soft. The volume of the supernatant is about 4 ml. If the volume of the liquid is obviously insufficient due to multiple precipitates, make up the volume with Teak Extraction Buffer.3) Prolonged incubation (up to 30 min) with potassium acetate will improve the quality of the RNA, especially in the presence of protein contamination. Polysaccharides form a grayish-white gelatinous precipitate. If the polysaccharide content of the tissue used is high. Extended incubation time (up to 60 min) also helps to precipitate more polysaccharides.4) No precipitate should be visible after the addition of isopropanol; any visible precipitate is indicative of the presence of large amounts of polysaccharides. Incubation times greater than 60 min will result in the formation of a polysaccharide precipitate.5) The RNA precipitate should be white, if there is a grayish-white gelatinous precipitate it is contaminated with polysaccharides. In this case, resuspend the precipitate in 200ul TE, add 1x volume of 2mol/L potassium acetate, and incubate on ice for 30 min. centrifuge at 12,000 g for 20 min at 4°C. Transfer the supernatant to a new 1.5 ml tube, and precipitate the RNA in 2.5x volume of 100% ethanol for 15 min at -20°C. Continue with step 16 in the method.)6) To determine the success of RNA isolation, the yield and quality of RNA can be assessed by measuring the UV absorption at 230nm, 260nm and 280mn. If A260/230value is less than 2, polysaccharide and/or polyphenol contamination is indicated; if A260/280If the A 260/280 ratio is less than 1. 7， it indicates protein contamination, and the presence of 28S and 18S rRNA in the agarose gel can be used to assess the integrity of the RNA.7) The RNA extracted by this method is suitable for poly(A)+ isolation, Northern blot analysis, cDNA analysis and RT-PCR amplification. For more product details, please visit Aladdin Scientific website.
Mortar and pestle Centrifuge Glass Pasteur pipette
1) 0.75 mol/L sodium citrate, pH 7.0 (containing citric acid), treated with 0.1% DEPC and autoclaved
2) 10% (m/V) N-lauroyl sarcosine (N-lauroyl sarcosine), 0.1% DEPC and autoclaved.
3) Thiocyanoguanidine buffer: 293 ml of sterile deionized water, 17.6 ml of 0.75mol/L sodium citrate, 26.4m 丨 10% N-dodecanol sarcosine, in the manufacturer's vials in the dissolution of 250 g of thiocyanocyanoguanidine (do not weigh)
4) Extraction buffer: every 5 ml of guanidine thiocyanate buffer plus 36ul of mercaptoethanol. If the extracted tissue contains polyphenols, the extraction buffer can be added to polyvinylpolypyrrolidone (polyvinypoiypyrrolidone) (20%, m/m).
5) Chloroform: isoamyl alcohol 49:1 (V/V)
6) 2mol/L sodium acetate with acetic acid to pH4.0
7) Acid phenol: 500 g of crystalline phenol dissolved in 500 mli of ionized water, 50 ml each stored at -20°C, 4°C for one month.
8) Isolactone
9) 70% and 100% ethanol
10) 2mol/L potassium acetate, plus glacial acetic acid to pH 4.8
11)10mol/L LiCl
12)TNE buffer: 10 mmol/L Tris-HCl, pH 7.5, room temperature, 15 mmol/L NaCl, 1 mmol/LEDTA
13)TE buffer:l0 mmol/LTris-HCL PH7.5 1 mmol/LTEDTA
14) Mortar and pestle
15) liquid nitrogen
16) Centrifuge
17) Glass Pasteur pipettes, dry baked at 200°C for at least 3 hours
II Methods of operation
1) Grind 0.4 g of tissue to a fine powder in liquid nitrogen, do not allow the tissue to thaw.
2) During grinding, add 3.5 ml of extraction buffer and grind thoroughly. Transfer the homogenate to a 10 ml polypropylene tube = rinse the pestle and mortar with 1 ml Extraction Buffer and transfer to a polypropylene tube.
3) 23000 g, 4°C, centrifuge 20mim.
4) Transfer the hS suspension into a l0 ml polypropylene tube using a toasted glass pipette.
5) Add 0.4 ml 2mol/L sodium acetate and mix well. Add 4 ml phenol and mix well. Add 0.8 ml chloroform:isoamyl alcohol and mix well.
6) Place polypropylene tube on ice for 15 min.
7) Centrifuge as in step 3)
8) Transfer the upper suspension into a 30 ml polypropylene tube using a baked glass pipette, add the same volume of 2 mol/L potassium acetate and mix well.
9) Place the polypropylene tube on ice for at least 30 min.
10) Centrifuge at 44000 g at 4°C for 20 min.
11) Transfer the supernatant into a 15 ml polyethylene tube, add 0.6 to 1x the volume of 100% pre-cooled isopropanol, mix well, and place at -20°C for 45 min.
12) centrifuge at 27OOOg for 20 min at 4℃.
13) Gently pour out the supernatant and wash the precipitate with Iml70¾ ethanol, centrifuge for 3mim as in step 12 to remove as much ethanol as possible.
14) Add 400ul of DEPC water to dissolve the precipitate and transfer to a 1.5 ml tube.
15) Add 100ul of 10mol/LLiCl and let stand for at least 2 hours.
16) Centrifuge at 12000 g for 20 min.
17) Wash the precipitate twice with 1 ml of 70% ethanol.
18) Add 200ul of TNE to resuspend the precipitate, then add 500ul of 100% ethanol and leave at -20°C for at least 15 min.
19) Centrifuge for 5 mim as in step 16.
20) Wash the precipitate twice with lml of 70% ethanol.
21) Add 200~400ul of TE to resuspend the precipitate.
22) Dilute each sample 30~50 times and measure the light absorption value at 230mn, 260mn, 280nm.