In vitro splicing analysis experiments in mammalian cells
In vitro splicing analysis experiments in mammalian cells
It is typical for splicing reactions to use nuclear extracts, i.e., S100 extracts complementing partially purified fractions of SR proteins or crude extracts, most often extracts from HeLa cells. Precursor mRNA substrates are usually prepared by in vitro transcription using phage polymerase. This experiment was derived from "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
In vitro splicing analysis experiments in mammalian cells
Principle
It is typical for splicing reactions to use nuclear extracts, i.e., S100 extracts complementing partially purified fractions of SR proteins or crude extracts, most often extracts from HeLa cells. Precursor mRNA substrates are usually prepared by in vitro transcription using phage polymerase.
Materials and Instruments
Precursor mRNA Substrate Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
ATP CP mixture HEPES-KOH polyvinyl alcohol splicing termination solution Tris-HCl saturated phenol RNA upsampling solution acrylamide-urea gel storage solution
All reagents should be prepared in high quality autoclaved water such as Milli Q (Millipore, Bedford, MA, USA) purified or double distilled water. All chemical reagents should be of ultrapure grade or a grade specifically designed for molecular biology preparations. Since in vitro splicing analysis involves RNA at the 1015 molar level, special care should be taken to avoid ribonuclease contamination.
1. Precursor mRNA Substrate
The substrate is prepared by in vitro transcription of a linearized plasmid, in which the target gene or a portion of it is cloned downstream of the phage promoter of the plasmid, usually using phage RNA polymerase isolated from SP6, T7 and T3 phages. Precursor mRNA containing a cap structure is more stable and more efficiently spliced than precursor mRNA molecules without caps. The most convenient and efficient way to prepare cap-containing precursor mRNA substrates is to transcribe them with dinucleotide primers ( 7mGpppG or GpppG). One, and in specific cases two or more, 32P-labeled nucleotides are usually used in the transcription reaction to homogenize the labeling of the precursor mRNA molecules synthesized by transcription. The transcribed precursor mRNA is extracted with phenol and purified by ethanol precipitation, and the yield and concentration of RNA is determined by radiolabeling, e.g., by trichloroacetic acid (TCA) precipitation. Since phage RNA polymerase is highly specific for its promoter, gel purification is usually not required after transcription. However, if a particular template produces heterogeneous transcripts, the full-length precursor mRNA should be purified by preparative denaturing polyacrylamide gel electrophoresis.
2. Reagents
(1) 25X ATP/CP mixture: 12.5 mmol/L ATP, 0.5 mol/L phosphocreatine (CP). Dispense each portion into a microcentrifuge tube and store at -20℃.
(2) 80 mmol/L high-purity MgCl2: 1 mol/L reserve solution, dilute to prepare working solution, dispense 0.2 ml per portion into microcentrifuge tubes and store at 4℃ or -20℃.
(3) 0.4 mol/L HEPES-KOH (pH 7.3), filtered through a 0.2 μm filter to remove bacteria and stored at 4°C. The working solution was prepared from a 1 mol/L reserve solution of MgCl2, diluted to prepare the working solution.
(4) 13% (m/V) polyvinyl alcohol ( PVA). Low molecular mass PVA (Signia, p-8136) should be used. To facilitate solubilization, suspend PVA in a capped glass vial with Miili-Q water and autoclave for 10 min. Dispense 1 ml per portion into microcentrifuge tubes and store at -20 °C.
(5) Splicing termination solution: 0.3 mol/L sodium acetate (pH 5.2), 0.1% (m/V) SDS, 62.5 μg/ml tRNA. store at room temperature or at 4 °C.
(6) Tris-HCl saturated phenol (pH 8.0).
(7) RNA Sampling Solution: 90% (V/V) formamide, 50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, 0.1% (V/V) bromophenol blue, 0.1% (m/V) xylene blue FF. Dispense 1 ml each in microcentrifuge tubes and store at -20°C.
(8) Acrylamide-urea gel storage solution: 19% (m/V) acrylamide, 1% (m/V) methacrylamide, 7 mol/L urea, 89 mmol/L Tris base, 89 mmol/L boric acid, and 2 mmol/L EDTA This stock solution was diluted with the same solution lacking only acrylamide to a working concentration (typically 4%-10% acrylamide), warmed to 37°C, and stored at -20 °C. The stock solution was then diluted to the working concentration (usually 4%-10% acrylamide) using the same solution. ), warmed to 37°C, and vacuum degassed before polymerization with ammonium persulfate (133 μl of 10% ammonium persulfate per 20 ml) and TEMED (10 μl per 20 ml). The concentration of acrylamide used was determined by the size of the desired splicing product. Higher concentrations of acrylamide resulted in slower migration of lasso RNA (lariat RNA, an intermediate in the RNA splicing process) molecules compared to longer linear precursor mRNA molecules, which facilitated the identification of the lassoed molecules in the intermediate and the product and increased the sensitivity, as the mobility of the lassoed molecules was higher than that observed in the prolonged radioautography exposure. This facilitates the identification of lasso molecules in intermediates and products and improves sensitivity because the migration rate of lasso molecules is slower than that of degraded RNA molecules observed in long radioautographic exposures.
Methods
1. In vitro splicing reaction
(1) Add freshly prepared splicing buffer mixture to a centrifuge tube on ice: 1.0 μl 25X ATP/CP mixture, 1.0 μl 80 mmoI/L MgCl2, 1.25 μl 0.4 mol/L HEPES-KOH (pH 7.3), 5.0 μl 13% PVA (added last), 20 fml (usually 0.1-0.4 μl) 32P-labeled precursor mRNA, and add Milli-Q water to 10 μl. 13% PVA (added last) was added to 10 μl. For 32P-labeled precursor mRNA, add Milli-Q water to 10 μl. 13% PVA is very viscous and should be added last and blown up and down with a P-20 pipette tip to gently mix and avoid foaming. Mix well and centrifuge instantly (~3 s).
(2) Thaw a sufficient amount of frozen HeLa cell nuclear splicing extract. Extracts are thawed at room temperature and immediately placed on ice; unused extracts can be stored at -70°C; repeated freezing and thawing does not generally result in loss of splicing activity.
(3) Splice reaction. Place the experimental centrifuge tube on ice and carefully aspirate 15 μl of Buffer D dialyzed HeLa cell nucleus extract (or S100 extract in Buffer D plus SR protein). Depending on the amount and concentration of extract, effective splicing usually requires either 5-10 μl of nuclear extract or 5-10 μl of S100 extract plus 15-20 pmol of SR protein. Add 10 μl of the splicing buffer mixture and gently mix all reagents using a new pipette tip and blowing up and down with a P-20 pipette tip (do not oscillate).
(4) Centrifuge instantaneously (~3 s) and incubate at 30°C for 1-4 h. Splicing efficiency depends on the specific precursor mRNA.
(5) Add 0.2 ml of splicing termination solution.
(6) Add 0.2 ml of Tris-saturated phenol and immediately shake for 1~2 min.
(7) Centrifuge for 5 min and transfer the aqueous phase to a new centrifuge tube. Avoid bringing in substances from the organic and intermediate phases to avoid contamination.
(8) Add 0.5 ml of ethanol and shake. Place on ice or freeze for at least 10 min and store overnight if desired.
2. Denaturing PAGE analysis of splice products
Smaller gels of 15-20cmX15-20cm are suitable for routine splicing analysis, but longer gels should be used if multiple RNAs of similar size are to be isolated. Thin gels less than 0.5 mm thick give sharper bands for autoradiography.
(1) Centrifuge the ethanol-precipitated RNA prepared in 1 "In Vitro Splicing Reaction" for 15 min, carefully removing the ethanol without stirring the precipitate. If the ethanol has been completely removed, drying is not necessary.
(2) Add 3~4 μl of RNA sampling solution and use a pipette to blow up and down or oscillate to dissolve the RNA precipitate.
(3) Heat at 80~85℃ for 5~10 min.
(4) The denaturing polyacrylamide gel should be pre-electrophoresed for at least 30 min prior to sample loading. wash the sample wells of the gel with a syringe to remove urea that has diffused into the wells prior to sampling, and load the heated samples directly (without cooling) into the individual wells.
(5) Electrophoresis until the indicator has migrated the desired distance.
(6) When the electrophoresis is complete, remove the electrophoresis unit and place a used X-ray slide or 3 MM paper on top of the gel and apply gentle pressure so that the gel adheres firmly to it. Turn the slide over and slowly pick up the slide from one side to make sure that the gel sticks to the film or paper. Cover the gel with a clean X-ray film.
(7) Radiographic autoradiography is usually performed at -70°C with a sensitizing screen. Exposure times usually range from 2 h to overnight, depending on the labeling activity of the RNA.