Section III Plant Virus RNA Extraction
Most of the plant viral RNAs are single-stranded RNA, and their polarity is the same as that of mRNA. The extraction of plant virus RNA is relatively simple, and phenol chloroform is generally used to obtain satisfactory results.
First, the material
The TMV virus solution (10 mg/ml) was purified.
Second, the equipment
Freezer bench centrifuge, low temperature vacuum dryer, electrophoresis, electrophoresis tank.
Third, the reagent
TE-saturated phenol: chloroform (1:1), chloroform, 3M NaAc (pH 5.2), ethanol (100% and 70%), TE buffer, double bacteria water without RNase.
Fourth, the operation steps
1. Add an eppendorf tube to the purified TMV (10 mg/ml) 400 ml, add an equal volume of phenol/chloroform, cover the tube cover, shake well for 1 minute, and centrifuge at 12000 g for 10 minutes at 4 °C.
2. Pipette the aqueous phase in a new eppendorf tube and extract with phenol/chloroform until there is no protein at the interface between the aqueous phase and the organic phase.
3. Pipette the aqueous phase into the new eppendorf heart tube, add an equal volume of chloroform, invert the tube by hand for several tens of seconds, and centrifuge at 12000 g for 10 minutes at 4 °C.
4. Take the aqueous phase, add 1/10 volume of 3 mol/L NaAc (pH 5.2), 2.5 volumes of ice-cold ethanol, mix, and -20 ° C for 30 minutes.
5. Centrifuge at 12000 g for 15 minutes at 4 ° C, carefully discard the supernatant, wash the pellet with 70% ethanol, and centrifuge at 12000 g for 5 minutes at 4 °C.
6. Discard the supernatant carefully, dry it in a vacuum for 5 minutes or air dry for 10 minutes, and dissolve in RNase-free double bacteria water or TE buffer.
7. Take 10 ml for electrophoresis analysis and another 10 ml for cDNA synthesis.
[Note] 1. The whole operation should be carried out at low temperature as much as possible.
2. Since the viral RNA is embedded in the coat protein, it is necessary to fully strip the viral coat protein, and it is generally necessary to perform phenol/chloroform extraction multiple times.
cDNA synthesis
Promega's RibocloneR M-MLV(H-) cDNA synthesis system replaces AMV reverse transcriptase with an RNase H deletion mutant of M-MLV reverse transcriptase to make the synthesized cDNA longer. The first strand of the system is synthesized using M-MLV reverse transcriptase, the second strand of cDNA is synthesized by displacement synthesis, RNaseH and DNA polymerase I are used for displacement synthesis, and finally the single-stranded end is cut with T4 DNA polymerase. Easy and convenient. The system reagents include:
20μg specific primer
200μl M-MLV first-chain buffer (5×), the formula is as follows: 250mmol/L Tris·Cl pH 8.3 (at 37°C); 375mmol/l KCl; 15mmol/L MgCl2; 50mmol/L DTT; 10mmol/L dATP, dCTP, dGTP, dTTP mixture (each 2.5mmol/L)
2×625μ rRNasinR RNase inhibitor
10,000μ M-MLV reverse transcriptase, RNase H-
5μg control RNA
400μl M-MLV second chain buffer (10×), the formula is as follows:
400mmol / L Tris · Cl, pH 7.2; 850mmol / L KCl; 44mmol / L MgCl2;
30 mmol/L DTT; 0.5 mg/ml BSA.
500μ RNase H
500μ DNA polymerase I
100μ T4 DNA polymerase I
2 x 1.25 ml nuclease-free water
All of the above reagents were stored at -70 ° C except for the control RNA, and the remaining reagents were stored at -20 ° C, and 40 μg of mRNA could be synthesized.
(1) First chain synthesis
Reagent
[α-32 P] dCTP (>400 Ci/mmol), EDTA (50 mM and 200 mM), TE-saturated phenol: chloroform (1:1), 7.5 M NH4Ac, ethanol (100% and 70%), TE buffer.
2. Operation steps
(1) Take a sterile RNase-free eppendorf tube, add the RNA template and appropriate primers, use 0.5 μg of primer per μg of RNA (using 0.3 μg using a NotI primer linker), and adjust the volume to 15 μl with H2O, 70 Treat at °C for 5 minutes, cool to room temperature, centrifuge to concentrate the solution on the bottom of the tube, then add sequentially
5× first strand buffer 5μl
rRNasin RNase inhibitor 25U
M-MLV(H- ) reverse transcriptase 200U
H 2 O adjusted to a total volume of 25μl
(2) Using a finger to flick the tube wall, pipette 5 μl into another eppendorf tube, and add 2-5 μCi [α-32 P] dCTP (>400 Ci/mmol) for the first-chain isotope incorporation of radioactivity assay.
(3) 37 ° C (random primer) or 42 ° C (other primers) warm bath for 1 hour
(4) Take out and put on ice
(5) The eppendorf tube incorporated into the assay was stopped by adding 95 μl of 50 mM EDTA, and the total volume was 100 μl. 90 μl of the electrophoresis analysis (first extraction with phenol) and another 10 μl of the isotope incorporation radioactivity assay were performed.
The first chain synthetic eppendorf tube can be directly used for second chain synthesis
Note: The amount of RNA used in the total volume of the above 25 μl reaction is 1 μg. If 5 μg of RNA is synthesized, the reaction volume can be scaled up, and 5 μg of RNA is synthesized using a total volume of 125 μl.
(ii) Second chain synthesis
1. Take 20 μl of the first strand reaction solution, and then add them in order.
10× second strand buffer 20μl
DNA polymerase I 23μ
RNase H 0.8μ
H2O is added to a final volume of 100μl
2. Mix gently. For the second strand isotope incorporation radioactivity assay, remove 10 μl into another eppendorf tube and add 2-5 μCi [α-32 P] dCTP.
3, 14 ° C warm bath for 2 hours (if you need to synthesize cDNA longer than 3 kb, you need to extend to 3-4 hours).
4. Add 90 μl of 50 mM EDTA to the eppendorf tube for incorporation, and take 10 μl for isotope incorporation radioactivity determination. The rest can be analyzed by electrophoresis.
5. The second strand of cDNA was synthesized and centrifuged at 70 ° C for 10 minutes, and placed on ice after low speed centrifugation.
6. Add 2μ T4 DNA polymerase and warm at 37 ° C for 10 minutes.
7. Stop the reaction by adding 10 μl of 200 mmol/L EDTA.
8. Extract the cDNA reaction solution with an equal volume of phenol:chloroform and centrifuge for 2 minutes.
9. Transfer the aqueous phase to another eppendorf tube, add 0.5 volume of 7.5M ammonium acetate (or 0.1 volume of 1.5 M sodium acetate, pH 5.2), mix and add 2.5 volumes of ice-cold ethanol (-20 °C), placed at -20 ° C for 30 minutes and then centrifuged for 5 minutes.
10. Carefully discard the supernatant, add 0.5 ml of ice-cold 70% ethanol, and centrifuge for 2 minutes.
11. Carefully remove the supernatant and dry the pellet.
12. The precipitate is dissolved in 10-20 μl of TE buffer.
(III) Determination, calculation and electrophoresis analysis of radioactive activity of isotope incorporation
Reagent
1 mg/ml salmon sperm DNA, trichloroacetic acid (TCA, 5% and 7%), alkaline agarose gel, alkaline gel running buffer, (30 mM NaOH, 1 mM EDTA), 2 x sample buffer, (20 mM NaOH) , 20% glycerol, 0.025% bromophenol blue).
2. Operation steps
(1) Take 3 μl of each of 2 (5) and 2 (4) reaction solutions, place on glass fiber filter paper, and dry at room temperature. These samples represent total radioactivity.
(2) Also take 3 μl of the reaction solution to 100 μl (1 mg/ml) of salmon sperm DNA, mix, add 0.5 ml of 5% TCA, mix with a vortex mixer, and place on ice for 5-30 minutes.
(3) Filter with glass fiber filter paper, wash three times with ice-cold 5% TCA, rinse with 5 ml of TCA each time, and then 5 ml of acetone or ethanol. These samples represent the incorporation of radioactivity.
(4) Determine the total radioactivity intensity and the intensity of the incorporated radioactivity, which can be counted by the Geiger calculator or by liquid flash.
3. First chain production determination
First chain incorporation rate (%) = incorporation of cpm / total cpm × 100%
Incorporation of dNTP (nmol) = 2 nmol dNTP / μl × reaction volume (μl) × (first chain incorporation rate)
Let 330 be the average molecular weight per mol dNTP
Amount of synthesized cDNA (ng) = incorporated into dNTP (nmol) × 330 ng / nmol
mRNA to cDNA conversion rate = synthetic cDNA amount (ng) / template RNA amount (ng) × 100%
For example, the total radioactive intensity is 254,000 cpm, the radioactive intensity is 3040 cpm, the amount of RNA used is 1 μg, and the reaction volume is 25 μl.
Incorporation rate = 3040/254000 × 100% = 1.2
Incorporation of dNTP = 2 nmol dNTP / μl × 25 × 1.2% = 0.6 nmol
Synthetic cDNA amount = 0.6 nmol dNTP × 330 ng / nmol = 198 ng
mRNA to cDNA conversion rate = 198 nm / 1000 ng × 100% = 19.8%
Since 20% (5 μl / 25 μl) of 1000 ng of RNA was used for the incorporation assay, and the reaction volume accounted for 80% of the total volume, the actual first strand cDNA synthesis amount.
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