There are two strategies for in vitro experiments; one is to co-incubate a plasmid containing the target gene at the Po ly( A) locus with cell-free extracts to allow RNA polymerase II to transcribe the target gene and process the initial transcription, and the other is to synthesize the pre-mRNA using a standard phage polymerase-driven system, and then co-incubate it with cell-free extracts.
Operation method
mRNA 3' end processing in vitro
Materials and Instruments
HcLa cells in logarithmic growth phase RNA polymerase relative to the promoter used Yeast tRNA Ultrastructured salmon sperm DNA S1 nuclease KlenowDNA polymerase Move -Materials and equipment Caveat 1) Regeneration of oligo dT cellulose: first rinse with water, then wash with 0.lmol/LNaOH and 5 mmol/LEDTA, and finally wash with water to remove NaOH thoroughly (pH test paper to determine pH). Oligomeric dT cellulose can be used 3 times.2) If RNA is not treated with an RNase inhibitor (e.g. DEPC), autoclaving will usually work as long as care is taken during handling. In addition, the pipettes used to manipulate the RNA should be specialized.3) The key to preparing cell-active extracts is good cell growth and rapid handling.4) There are two critical steps in the S1 nuclease analysis that must be taken at all times to ensure that the temperature of the hybridization mixture does not fall below the hybridization temperature. These two steps are: firstly, the reaction tube must be transferred quickly from the denaturation step to the hybridization bath, and secondly, the reaction tube must remain in the hybridization bath when the digestion mixture is added to the reaction plants. The optimal hybridization temperature produces clean signals with low background. For most probes, 55-60°C is suitable, but for some probes, the hybridization temperature needs to be adjusted according to the specific situation.5) If you are testing only the cleavage of pre-RNA, add 0.5 mmol/L 3'ATP to the reaction system and incorporate it into the cleavage site in the presence of pdy(A)+ polymerase in the extract, causing chain termination. For more product details, please visit Aladdin Scientific website.
MWCO3500 (1 ml cm) dialysis bag Dounce homogenizer Gorrex tubing 15 mlFalcon or Corning tubing Econo chromatography columns X-ray film and exposure kit
(i) Nuclear extract preparation
1) HcLa cells in logarithmic growth phase.
2) PBS: 0.2 g/L KCl , 0.2 g/L KH2P04, 8.0 g/L NaCl2., 16 g/L Na2HPO4.7 H20
The following buffers A, C, and D were stored at 4°C. DTT was prepared as a 1 mol/L storage solution and diluted as it was used.
3) Buffer A: 10 mmol/LHEPES, pH7.9 at 4℃, 10 mmol/LKCl, 1.5 mmol/L MgCl2. 0.5 mmol/LDTT.
4) Buffer C: 20 mmol/LHEPES, pH7.9 at 4°C, 420 mmol/LNaCl, 0.2 mmol/LEDTA, 25% glycerol (USP grade), 1.5 mmol/LMgCL2, 0.5 mmol/LDTT.
5) Buffer D: 20 mmol/L,HEPES, pH 7.9 at 4°C .l00 mmol/LKC1, 0.2 mmo1/L EDTA, 20% glycerol, 0.5 mmol/LDTT.
6) MWCO 3500 (1 ml/cm) dialysis bag (Spectropre)
7)0.04% Taipan Blue
8)Dounce homogenizer
9)Gorrex tube
10) 15 ml Falcon or Corning centrifuge tube.
(ii) Pre-mRNA preparation
1) Reaction buffer: 40 mmol/L Tris-HCl (pH 8.0), 25 mmol/L NaCl, 8 mmol/L MgCl2, 2 mmol/L spermidine.
2) 280 mmol/L cap analog (m' G[5']ppp[5']G, Pharmacia.Picataway, NJ,USA).
3] 1moL/LDTT.
4] 1Ommol/LGTP
5)100 mmol/LUTP,ATP,CTP solution
6) [5 H]CTP, 30-60Ci/mmol
7) RNA polymerase relative to the promoter used.
8) DNase without RMase
(iii) In vitro processing
1) 50 mmol/LMgCl2
2)10% polyvinyl alcohol.
3)0.5 mol/L phosphocreatine.
4)10 mmol/L four GTP, ATP, UTF, CTP
5)25 mmol/L ATP
6) Final buffer: 50 mmol/L Tris-HCl (pH 7.4) 50 mmol/L EDTA, O.l% SDS
7) Protease K
S) 10 mmol/LTris-HCl(pH7,4),10 mmoL/LEDTA saturated phenol
9) Chloroform: isoamyl alcohol (24:1)
10) 1.5 mol/LNH4Ac
11)10 mg/ml yeast tRNA
12) Ethanol
(iv) Oligomeric dT cellulose chromatography
1) 5mol/LNaCL
2) Oligomeric dT cellulose.
3) TE: 10 mmol/LTris-HCl(PH7.4), lmmol/L EDTA
4) Binding buffer: TE ten 0.5 mol/LNaCl
5) Wash buffer: TE+0.1mol/LNaCl
6) 3mol./LNaAc
7) Econo chromatography column (Bio-Rad,Hercules,CA,USA) or other primary chromatography device.
(v) S1 Nuclease analysis
1) S1 hybridization buffer: 80% ion ultra-pure formamide, lOmmol/LPIPES (pH 6.1), 0.4mol/LNaCl
2) 10mol/ml of salmon sperm DNA broken by ultrasonics.
3)5XS1 digestion buffer:0.15mol/LNaAc(pH4.5),2mol/LNaCl,5 mmol/LZnSO4
4)S1 Nuclease.
5)40% acrylamide solution (acrylamide: bis(methylenedienyl)amide 19:1)
S) Gel loading buffer: 80% deionized formamide, 0.1% bromophenol blue, 0.1% xylene blue, lOmmol/L NaOH, 1XTBE
7) 10XTBE: 108 g/L Tris base, 55 g/L boric acid, 9.3 g/L Na2EDTA.
8) X-ray film and exposure cassette. If the results need to be correlated, a densitometer or other quantitative measurement equipment is required.
9) [32P]dNTPT 3000Ci/mmol, the dNTP used is determined by the sequence of the probe.
1) Klenow nDNA polymerase
II. Methods of operation
(1) Preparation of nuclear extract
Nuclear extracts are usually prepared using HeLa cells, but other cell lines may also be used. The key to preparing effective nuclear extracts is to harvest the cells during the logarithmic growth phase, and to operate them at low temperature for as short a time as possible. Transflasks can generally be used to culture the cells so that the most human extracts can be obtained, and the same method of manipulation described below applies to cells in adherent wall cultures.
1) Harvest cells by physical or tryptic digestion, centrifuge at 200 g at 4°C and wash cells 3 times with pre-cooled sterilized PBS.
2) Resuspend the cells in 5x volume of Buffer A (note the addition of DTT) (note that this step and subsequent operations should be performed at low temperature). After the cells have been fully suspended and placed on ice for 10 min, transfer the cells to pre-cooled, sterilized Correx tubes and centrifuge at 600 g at 4°C for 1 minute. The volume of the precipitated cells should have expanded by this time, remove the supernatant and add 2x the volume of Buffer A to fully suspend the cells.
(3) Transfer the mixed cells to a pre-cooled Dounce homogenizer and homogenize for about 15 times. Take a small amount of sample and mix it with an equal amount of Taipan blue to determine the cell lysis, and observe it under a microscope. The nuclei of lysed cell plants take up the dye and turn blue, while intact cells are colorless. It is important to ensure that more than 90% of the cells are lysed. The lysed cells are transferred to pre-cooled, sterilized Correx tubes and centrifuged at 1500 g for lOmin at 4°C to collect the nuclei.
4) After centrifugation of the nuclei, remove the supernatant and add 3 ml of Buffer C per IX109 cells to the starting cell volume, the amount of Buffer C added is used as a constant key.
5) Suspend the precipitate well, transfer the nuclei to a homogenizer and homogenize 15 times, then transfer the nuclei to a pre-cooled and sterilized Falcon tube and gently mix at 4°C for 1 hour on a rotator.
6) Wash the dialysis bag with distilled water and immerse it in distilled water. At the same time, add 1L of buffer D (note the addition of DTT) to a sterilized 2L beaker with a sterilized stirrer and place it at 4°C.
7) After the extraction step, the solution containing the nuclei was transferred into pre-cooled sterilized Correx tubes and centrifuged at 10,000 g for 30 min at 4°C. The upper part of the sample was transferred into a dialysis bag, tightly closed, and placed in Buffer D. The plants were dialyzed in a cold room for 2-2.5 h. Afterwards, the samples were dispensed into micro-centrifuge tubes on ice with dry ice and were immediately stored in a 70°C room. Protein concentration was determined by conventional methods.
(ii) Pre-synthesis of mRNA by phage RNA polymerase
The plasmid for pre-mRNA preparation contains no initiating T and encoding poly(A)+ site that can be recognized by phage RNA polymerases such as T7, T3, SP6, etc. The plasmid is cleaved by restriction endonuclease at the 3' end and downstream of the signal to form a linear DNA, which is then extracted with phenol and chloroform, purified by ethanol precipitation, and used in the transcription reaction. The [32P]UTP used in the synthesis of RNA allows direct detection of the dove product; if [32P ]-labeled nucleotides are not used, a [32P]-labeled S1 probe can be used. The following method prepares pre-mRNA using [3 H] tracer labeling, and its subsequent processing can be measured using S1 nuclease analysis.
1) Add 2ug of linearin to the reaction system containing 40 mmol/L Tris (pH 8.0), 25 mmol/L NaCl, 2 mmol/L MgCl2, 2 mmol/L semicarboxamidine, 2.8 mmol/L cap analog, 5 mmol/LDTT, 5uCi[3 H]UTP, 100 mol/L GTF, lmmol/L ATP, UTP, and CTP. The reaction system was incubated at 18 ℃ with 2ug of linear DNA template and T7RNA polymerase, and finally 2U of RNase-free DNase was added and the reaction was incubated at 37 ℃ for 15 min to terminate the reaction.
2) The RNA yield can be determined from the amount of UTp incorporation into the TCA precipitates, and the processing of this pre-mRNA can be determined by hybridization with a 3' end-labeled DNA probe, so it is not necessary to purify the full-length molecule.
(iii) Pre-mRNA processing reaction
1) Add 2nmol/L(l00fmol) of pre-mRNA into a 50ul reaction system containing 2 mmol/LMgCl2, 3% polyvinyl alcohol, 20 mmol/L phosphocreatine ATP, and 1nmol/L(l00fmol) of nuclear extract (the amount of nuclear extract should be measured each time, and 100ug is usually enough); the amount of pre-mRNA should be measured as well, and the ideal amount is 1-10nmmol/L). The amount of pre-mRNA should also be measured, and the ideal amount is 1-10 nmmol/L)
2) Incubate at 30°C for 2 h
3) Add equal amount of termination buffer and 30ug of proteinase K, incubate at 37℃ for 15 min to terminate the reaction. Extract the RNA once with phenol, once with chloroform/isoamyl alcohol, and then add ammonium acetate, 25ug yeast tRNA and 2 times the volume of ethanol to precipitate the RNA.
4) When [32P ]-labeled RNA is used, the product is determined by gel electrophoresis, and if [3 H]UTP-labeled RNA is used, the RNA product is purified by oligo-dT cellulose chromatography prior to S1 analysis.
(iv) Transcription processing reactions
1) Incubate 1ug of superhelical DNA substrate with 100ug of nuclear extract, 3.0% polyvinyl alcohol, 20 mmol/L phosphocreatine, 2 mmol/L MgCl2, 600 mmol/L each of GTF, ATP, UTP, and CTP in a 501-ul reaction system at 30°C for 2 h. Incubate at 30°C for 2 h.
2) Add equal amount of termination buffer and 30ug of proteinase K. Incubate at 37℃ to terminate the reaction. Extract the RNA once with phenol, chloroform/isoamyl alcohol, then add 2.5mol/L ammonium acetate, 25ug yeast tRNA and 2 times volume of ethanol to precipitate the RNA, and then purify Poly(A)+RNA by oligo dT cellulose column.
(E) Oligomeric dT cellulose column chromatography
1) Fill an oligo dT cellulose column with 0.lml of column bed volume and equilibrate the column with 5 times the volume of binding buffer.
2) Dissolve RNA in TE, heat to 60℃, denature for 5 min and place on ice, then add NaCl to the system plants to a final concentration of 0.5mol/L, to prevent RNA denaturation, load the RNA sample onto the column, collect the effluent, and then load the column again.
3) Wash the pestle and mortar with 5 times the volume of binding buffer, collect the effluent, and add the effluent from the sampling process. This is the Poly(A)+ fraction, which can be discarded or used as a backup.
4) Wash the column with 3x volume of TE containing 0.1moL/L NaCL and discard the eluate.
5) Elute Poly(A)+ RNA with 3x volume of TE and collect the eluate.
6) Precipitate RNA with a final concentration of 0.3 mol/L NaAc and 2x volume of ethanol.
(vi) S1 Nuclease analysis of processed RNA
1) Cut the plasmid upstream of the poly A site with restriction endonuclease to form a protruding end and use it as a DNA probe for S1 analysis. The ends are flattened with KlenowDNA polymerase and the corresponding [32P]dNTP, and the DNA is purified prior to the flattening reaction, but for most digestion reactions, 50-lOOuCi [ a-32P ] radiolabeled ribonucleic acid (3,000 Ci/mmol) and 1UKlenow enzyme can be added directly after digestion and incubated on ice for 30 min. After labeling the reaction, the DNA is extracted with phenol and chloroform. After the labeling reaction, the DNA was extracted with phenol and chloroform, precipitated with ethanol, and cleaved downstream of the poly A site with a second enzyme, and the labeled fragments were purified using conventional gels.
2) Dissolve the precipitation probe and RNA in 150ul of S1 hybridization buffer. The amount of probe must be judged empirically to be sufficient to ensure that the amount of probe exceeds the amount of product in the reaction. Heat the mixture at 90-95°C and quickly immerse the tubes in a water bath at the appropriate temperature, which must be done quickly to ensure that the temperature never falls below the hybridization temperature, and hybridize overnight.
3) Prepare the following mixture on ice: 100ul of 5×S1 Clostridium Digestion Buffer, 35ul of redistilled water, 10ug of freshly denatured (boiled for 5 min) sonically interrupted salmon sperm DNA, and 250US1 nuclease (this amount of S1 nuclease is usually sufficient to complete the reaction but should be adjusted appropriately based on experience). Before the digestion is initiated, carefully open the reaction tube containing the hybridization mixture. Before digestion begins, carefully open the reaction tube containing the hybridization mixture, keep the tube on ice at all times, add 150ul of ice pre-cooled digestion mixture to the tube, immediately remove the tube from the water bath, cap it, turn it upside down several times to mix, and place it back on ice. All tubes are processed until all tubes have been processed, then centrifuged quickly and briefly, and the tubes are again transferred to a water bath at 20-25°C for digestion. If the AT content of the hybridization sequence is high, the temperature can also be slightly lower. Digestion is usually completed within 1-2 h, but can be adjusted for different probes based on experience.
(vii) Electrophoretic analysis of reaction products
When using polyacrylamide urea sequencing gel to isolate labeled RNA or the product obtained by S1 nuclease analysis, when using isotopically labeled RNA inches, there will be bands formed by the sample RNA on the gel as well as slower migrating ladder bands that are suitable for cleavage and polyadenylation of the product, the formation of the ladder bands is due to the different lengths of the poly(A)+ tails. If the RNA plus pdy(A)+ tail reaction is inhibited, the 5' end excision is observed on the gel. It is observed that the 5' end excision product migrates faster than the added RNA. During S1 nuclease analysis. Undigested probes as well as digested products can be detected due to the protective effect of the cleaved RNA.