RNA Laboratory Techniques Manual (Molecular Cloning - Laboratory Guide Series)
Operation method
Isolation of mitochondrial RNA from leaves of green leafy plants
Principle
Using differential centrifugation, mitochondria were separated from other subcellular structures and further purified by sucrose gradient centrifugation to obtain mitochondria. Chloroplast RNA was removed from the mitochondria by treatment with ribonuclease A. The mitochondrial RNA was inactivated by the addition of a high concentration of guanidine thiocyanate, which is very effective as a protein denaturant, and the mitochondrial RNA was precipitated by centrifugation in a CsCl gradient. The mitochondrial RNA was precipitated by CsCl gradient centrifugation, and the single-stranded DNA precipitated by CsCl gradient centrifugation was removed from the mitochondrial RNA by LiCl precipitation.
Materials and Instruments
5.7moI LCsCl solution 10 mmol LEDTA pH7.5 DEPC water Denaturing buffer EB extraction buffer 4mol L Guanidine thiocyanate 7.5mol L Guanidine hydrochloride 2mol L and 4mcL LLiCl Sampling buffer 10XMOPS buffer 2mol L Potassium acetate 2mol L Sodium acetate 5% Sodium dodecyl sarcosinate TE buffer Elution Buffer Move I. Materials and equipment Caveat 1) Wear plastic or latex gloves during the operation. All reagents were used only for KNA extraction to avoid the contamination of ribonuclease. All glassware should be treated at 160°C for 4 h. Plasticware used should be treated with guanidine thiocyanate and immersed in 0.2% DEPC water for 12 h and sterilized by autoclaving.2) The gradient solution prepared for sucrose gradient centrifugation should be equilibrated at 4°C overnight. After gradient centrifugation, elution buffer is slowly added to the collected mitochondria (15-20 mim), which reduces osmotic shock.3) Since single-stranded DNA and RNA are precipitated simultaneously in the gradient, 2moL/LiC1 precipitation is necessary to obtain purified RNA.4) Pure formamide reagent can be used directly. If yellow color appears, treat with 5% (m/V) resin 501-X8(D) (BioRad) for lh to remove ions. Filtered deionized formamide should be stored at -70°C. For more product details, please visit Aladdin Scientific website.
Homogenizer
The solutions were prepared with DEPC water:
1) 5.7moI/LCsCl solution.
2)10 mmol/LEDTA,pH7.5
3)DEPC water
4) Denaturation buffer: 50% formamide, 12% formaldehyde, IXMOPS buffer (40 mmol/LMOPS, 10 mmol/L sodium acetate, lmmol/LEDTA, pH 7.0), freshly mixed before use.
5) lmg/mlEB
6) Extraction buffer: 0.35 mol/L sorbitol, 50 mmol/L Tris-HCl(pH8.0).5 mmol/L LEDTA, 0.1% BSA,0.25 mg/ml spermine and spermidine, stored with mercaptoethanol to a final concentration of 0.2% before use.
7) 4mol/L guanidine thiocyanate: 100 mmoI/LTris-HCl (pH 7.5), add mercaptoethanol to 1% (final concentration) before use, store at 4°C.
8)7.5mol/L guanidine hydrochloride: containing 10 mmol/LDTT, NaOH adjusted to pH7.5, filtration and storage at 4℃.
9)2mol/L and 4mcL/LLiCl, store at 4°C.
10) Sampling buffer: 50% glycerol, 0.25% bromophenol blue, lmmol/LDTA.
11) 10XMOPS buffer: 0.4moI/LMOPS, 0.lmol/L. sodium acetate, 10 mmol/LNa2EDTA, pH 7.0.
12)2mol/L potassium acetate: pH5.
13)2mol/L sodium acetate: pH 7.5.
14)5% sodium dodecyl sarcosinate.
15)TE buffer: 10 mmol/L-Tris-HCl (pH 8.0), 1 mmol/LEDTA
16) Elution buffer: 350 mmol/L sorbitol, 50 mmol/LTris-HCl (pH 8.0), 20 mmol/LEDTA
17) Screeder
II Methods of operation
1. Isolation of mitochondria
All steps were carried out at ℃. Solution. Reagent bottles, etc. should be placed on ice.
1) Collect 20 g of fresh green leaves from oilseed rape or other plants 4 to 6 weeks old. Cut and cool in 200 ml of ice-cold extraction buffer.
2) Homogenize the tissue three times in a homogenizer at island speed [l0S between each homogenization). Filter the homogenate through two layers of filter membrane into a 250 ml cold centrifuge bottle.
3) Centrifuge the filtrate at 2000 g for lOmin. Carefully transfer the supernatant to a new tube and centrifuge at 10,000 g for 20 min.
4) Resuspend the precipitate in 100 ml Teak Extraction Buffer and repeat step 3) - again.
5) Resuspend the mitochondrial precipitate in 20 ml of cold elution buffer.
6) Place each 10 ml of mitochondrial suspension carefully on top of a sucrose gradient (sucrose gradient consisting of 9 ml 0.9 mol/L, 11 ml 1.5 mol/L, 9 ml 1.75 mol/L, sucrose dissolved in elution buffer) and centrifuge at 80,000 g for 60 min. Collect the mitochondria from the interface of 0.9 mol/L and 1.5 mol/L sucrose lysine (yellow bands) using a wide bore pipette. Collect mitochondria from the 0.9mol/L and 1.5mol/L sucrose-lined interface (yellow band) using a wide-bore pipette and dilute with 5-fold elution buffer for 15-20 min
7) The mitochondria were precipitated by centrifugation at 10000 g for 20mim. The mitochondrial precipitate was resuspended in 1 ml of cold elution buffer.
2. Mitochondrial RNA isolation
1) Incubate mitochondria with 20ug/ml Ribonuclease A on ice for 60 min.
2) Add 5 times the volume of 4mol/L guanidine thiocyanate to the mitochondria. 0.5 times the volume of 5% sodium dodecyl sarcosinate is added after 60s at room temperature and mixed well. 5000 g, 5 min centrifugation of the mixture is performed to remove insoluble residue.
3) Each 3.2 ml of the mixture is placed in 1.lml of DEPC-treated 5.7mol/LCsCl solution.
4) Hypercentrifuge at 22,000 g for 14 hours.
5) Carefully aspirate the supernatant and excise the upper part of the centrifuge tube in contact with the homogenate (all steps should be taken to avoid contamination with Ribonuclease A on hand)
6) Add lml of 7.5mol/L guanidine hydrochloride and mix well to dissolve the RNA precipitate.
7) Add 0.05 times the volume of 2mol/L potassium acetate (pH 5.5) and 0.5 times the volume of ethanol to the mixture.
8) Incubate at ~20℃ for 4 h. Centrifuge at 5000 g for lOmin to precipitate mitochondrial RNA.
9) Add 0.1 times the volume of 2 mol/L sodium acetate (pH 7.0) and 2.5 times the volume of ethanol, incubate at -20°C overnight, and centrifuge at 10,000 g for 30 min.
10) Wash mitochondrial RNA with 70% ethanol, vacuum dry, and dissolve in 0.5 mlTE.
11) To obtain mitochondrial RNA without single-stranded DNA, dissolve the RNA by adding an equal volume of 4mol/LiCl, incubate at 4°C overnight, and centrifuge at 10,000 g for 20 min. Wash the mitochondrial RNA once with 2mol/LiC1 and twice with 70% ethanol.
12) Vacuum dry mitochondrial RNA and dissolve in DEPC-treated water. Measure the absorbance at 260 nm to calculate the yield of mitochondrial RNA. n
13) For long term storage, add 0.3 times the volume of sodium acetate and 2.5 times the volume of anhydrous ethanol to the mitochondrial RNA, and store at 0.3 to 0.5ug of mitochondrial RNA per gram of fresh leaves.