RNA Laboratory Techniques Manual (Molecular Cloning - Laboratory Guide Series)
Operation method
Hepatitis A virus RNA extraction
Materials and Instruments
TSES Buffer TSES Buffer Saturated Phenol Chloroform Octanol Potassium Acetate Ethanol Buffer A lmol LNaH2P04 Buffer Chloramine T Solution Na125I 5 mmol LNa2S2O5 lOOmmol LKI NET Buffer Yeast tRNA Sucrose Cellulose Column Equilibrium Triple Distilled Water 0.1 mol LNaOH 5 mmol LEDTA 0.1 mol LNaCL TES buffer 3 mol LNaAc formamide Move I Materials and equipment For more product details, please visit Aladdin Scientific website.
SephadexG-25 column Oligodeoxythymidine (digo-dT) cellulose column Polyuric acid column
1) TSES buffer: 0.2mol/LTris-HCl, 0.05mol/LNaCl0.011mol/LEDTA,0.5% SDS
2)TSES buffer saturated phenol
3) Chloroform:Octanol (24:1)
4) Potassium acetate
5) Ethanol
6) Buffer A: 0.lmol/LTris-HCl (pH 7.4), 50 mmol/LNaCl10 mmol/LEDTA, 0.2% SDS
7) lmol/LNaH2P04 buffer, pH 7.4
8) Chloramine T solution: 100ug of chloramine T dissolved in 25ul of 50 mmol/LNaH2P04 buffer.
9) Na125I
10)5 mmol/LNa2S2O5
11)l00 mmol/LKI
12)SephadexG-25 column
13)NET buffer: 10 mmol/LTris-HCl(pH7.4), 100 mmol/LNaCllOmmol/LEDTA
14) Yeast tRNA
15) Sucrose
16) oligodeoxythymidine (digo-dT) cellulose columns
17) Cellulose column equilibrium solution: 0.5 mol/L NaCl, 1 mmol/L EDTA, 20 mmol/L Tris-HCl (pH 7.6), 0.1% SDS
18) Triple distilled water
19) 0.1 mol/LNaOH
20) 5 mmol/LEDTA
21)0.1mol/LNaCL
22)TES buffer: 10 mmol/LTris-HCl (pH 7.5), lmmol/LEDTA, 0.05% SDS
23)3mol/LNaAc
24) polyuridylic-acid-Sepharose4B column (polyuridylic-acid-Sepharose)
25) Formamide
Methods of operation
1. Phenol: chloroform: octanol method of whole RNA extraction and purification
1) Add a volume of TSES buffer to the purified Hepatitis A virus (HAV).
2) Vibrate for 30s to 1 min and place in an ice bath for 15 min.
3) Add 1/2 volume of TSES buffer saturated with phenol, then add 1/2 volume of chloroform:octanol, and shake the mixture for 5~lOmin.
4) Centrifuge at 2500r/min at 4°C for 20 min.
5) Aspirate the aqueous phase and add 1/2 volume of TSHS buffer saturated with phenol and 1/2 volume of chloroform:octanol and repeat the extraction 4 times as above. The aqueous phase is the whole nucleic acid.
6) Add 2% potassium acetate and 2 times the volume of cold ethanol to the whole nucleic acid and place at -20°C for 2 h. Remove flocculent (DNA) by stirring gently with a fine glass rod or a long hypodermic needle.
7) Centrifuge at 4000r/min at 4°C for 20 min.
8) Discard the supernatant and re-dissolve the precipitate in the original volume of buffer A
9) Add proteinase K to a final concentration of 200ug/ml and incubate for 1~2 h at 37°C.
10) TSES saturated phenol: chloroform: octanol extraction once more. Centrifuge at 4000r/mim for 20 min at 4℃.
11) Dissolve the precipitate in the original volume of buffer A, add 2.5 times the volume of cold ethanol, and incubate for 2 h at 20℃.
12) Centrifuge at 10,000r/min for 30 min, precipitate is whole RNA.
2. Chloramine T-sucrose density gradient centrifugation for whole RNA purification.
1) TSES saturated phenol: chloroform: octanol extraction 4 times can obtain the whole RNA of HAV
2) Add 10ul of 1mol/LNaH2PO4 buffer, 10ul of chloramine T solution, and 15ul of Na125I if iodine labeling is required, to the whole RNA extract.
3) Shake the mixture at 25℃ for 25 min.
4) Add 100ul of 5 mmol/LNa2S2O5 and 200ul of 100 mmol/LKI to terminate the reaction.
5) Remove iodide from the reaction product. The reaction product was subjected to SephadexG-25 column chromatography, and the elution buffer was NET
6) Precipitate the eluate with anhydrous ethanol and add 100ul of yeast tRNA as carrier.
7) Centrifuge at 10,000r/min for 30 min at 4℃.
8) Add ethanol to the precipitated plants and wash twice, centrifuge as in 7).
9) The precipitate is redissolved in NET buffer.
10) Sucrose density gradient ultracentrifugation, take 500ul of RNA sample and add 4 ml of 15% to 30% Sucrose Zen solution prepared with NET buffer, centrifuge at 7℃, 310000r/min for 2.33h. The sample was centrifuged at 310,000 r/min at 7°C for 2.33 h. After centrifugation, four peaks were seen, namely, 4S, 18S, 28S, and 35S. Enzyme-specificity experiments proved that the 35S fraction was insensitive to DNAase and sensitive to RNAase, and therefore, this fraction was proved to be HAVRNA.
3) Extraction of Poly(A )+RNA
After phenol:chloroform:octanol and density gradient ultracentrifugation as described above, the following method can be used to isolate poly(A)-containing HAVRNA from the collected 35SRNA fraction.
1) Oligodeoxythymidine (oligo-dT) cellulose column chromatography method. The bed of the column should be 0.5 cmX2 cm or lcmX4.5 cm, depending on the isolation halo. ② Equilibrate the cellulose with cellulose column equilibrium solution. The column was loaded, washed three times with sterile triple-distilled water, 0.lmol/L NaOH and 5 mmol/L EDTA, and finally washed with water, and then washed once with equilibrium solution containing 0.lmol/L NaCl. Add the sample, dilute the sample with sterile triple-distilled water, treat with a water bath for 5mim, cool, and add to the column. ⑤ Elute with 2~3 times the volume of TES buffer, collect the eluent, and elute until the A260<0.03. ⑥ Collect the A260 peak portion, add 3mol/LNaAc and 2.5 times the volume of ethanol, and place it at 20 ℃. ⑦ Centrifuge the sample at 4 ℃, 10,000r/min for 30 min. 30 min. ⑧ The precipitate was redissolved in water, OD value was measured, and the RNA content was calculated as halo, and the HAVRNA containing poly(A) was obtained.
2) Polyuridine nucleoside column (polyuridyncaad-Sepharcst-4B) chromatography method. The experimental method and procedure were basically the same as that of 1). The column bed was lcmX2 cm, and the equilibrium solution was 1 part of formamide and 3 parts of NET buffer, and the column was equilibrated and washed 5 times. The poly(A)RNA elution solution was 9 parts of formamide and 1 part of NET buffer, and the RNA elution solution was collected, precipitated, concentrated and dried, and the RNA samples should be stored at -70 ℃ for preparation.