In vivo isolation of ribonucleoprotein complex by immunoprecipitation-rPCR of RNA
In vivo isolation of ribonucleoprotein complex by immunoprecipitation-rPCR of RNA
The RNA Immunoprecipitation-Randomized Polymerization Chain Reaction method can be used to identify RNA components in intracellular RNP complexes. This experiment is from the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
In vivo isolation of ribonucleoprotein complex by immunoprecipitation-rPCR of RNA
Principle
The RNA Immunoprecipitation-Randomized Polymerization Chain Reaction method can be used to identify RNA components in intracellular RNP complexes.
Materials and Instruments
Carrier yeast tRNA RQ1 DNA enzyme Glycogen Calf Intestine Alkaline Phosphatase T4 DNA Ligase JMJ09 Receptor Bacteria Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
PBS NET-2 buffer RNase inhibitor Phenol Chloroform Isoamyl alcohol Sodium ethanol M-MLV Reverse transcriptase Reverse transcription buffer Universal primer-dN6 RNase H Electrophoresis buffer Non-denaturing agarose gel buffer EcoR Ⅰ restriction endonuclease EDTA
Protein A Agarose Gel RNA-free Enzyme Centrifuge Column PCR Amplification System Promega PCR Kit pGEM-7Z Plasmid Vector Falcon 2059 Polypropylene Tubes SOC Medium LB Plate Medium fmol DNA Cycle Sequencing System Microgel Horizontal Electrophoresis Ultraviolet Transilluminator PCR Amplification Instrument High-Speed Cryogenic Centrifuge Cryogenic Microcentrifuge Tabletop Cryogenic Centrifuge Spinning Vacuum Concentrator
1. Preparation of cell extracts
(1) Sterile 1X PBS.
(2) NET-2 buffer: 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.05% (V/V) NP-40.
(3) RNase inhibitor.
2. Immunoprecipitation
(1) Protein A agarose gel.
(2) Vector yeast tRNA (1 mg/ml).
(3) RNase inhibitor.
(4) NET-2 buffer.
(5) RQ1 DNA enzyme (Promega).
(6) Phenol: Chloroform ( 1 : 1 ), keep away from light.
(7) Chloroform: isoamyl alcohol ( 24:1 ).
(8) 3 mol/L sodium acetate (pH 5.2 ).
(9) Glycogen.
(10) Anhydrous ethanol and 80% ethanol.
(11) DEPC-treated water.
3. RNA-rPCR method
(1) M-MLV reverse transcriptase and reverse transcription buffer: 50 mmol/L Tris-HCl (pH 8.3), 10 mmol/L DTT, 3 mmol/L MgCl2, dNTP (0.5 mmol/L each), 1 U RNase inhibitor, 50 U M-MLV reverse transcriptase.
(2) Universal primer-dN6: 5' -GCCGCTCGAGTGCAGAATTCNNNNNNN-3'.
(3) RNase H.
(4) Second-strand cDNA synthesis: DNA polymerase I Klenow blockbuster, 10X DNA polymerase I Klenow blockbuster reaction buffer, dNTP mix (25 mmol/L each).
(5) Phenol: Chloroform (1 : 1), stored away from light.
(6) Chloroform: isoamyl alcohol (24 : 1).
(7) G-50, D-RF RNAase-free centrifugal columns (5-Prime, 3-Prime Inc.).
(8) PCR amplification system: Taq DNA polymerase, 10X PCR buffer [ 100 mmol/L Tris-HCl (pH 8.3), 500 mmol/L KCl, 15 mmol/L MgCl2, 0.1% gelatin], and dNTP mixture (10 mmol/ L each).
(9) Universal primer: 5'-GCCGCCGAGTGCAGAATTC-3'.
(10) DEPC-treated water.
(11) Electrophoresis buffer: 1X TBE.
(12) 10X non-denaturing agarose gel uploading buffer: 0.25% bromophenol blue, 0.25% xylene cyanide, 15% (m / V) Ficoll 400, dissolved in deionized water.
4. Construction of cDNA plasmid library and transformation
(1) Promega PCR kit.
(2) EcoR Ⅰ restriction endonuclease.
(3) pGEM-7Z plasmid vector (Promega).
(4) Calf intestine alkaline phosphatase and its 10X buffer.
(5) 0.5 mol/L EDTA.
(6) Phenol : Chloroform (1 : 1), keep away from light.
(7) Chloroform: isoamyl alcohol (24 : 1 ).
(8) 3 mol/L sodium acetate (pH 5.2 ).
(9) Anhydrous ethanol and 80% ethanol, stored at -20℃.
(10) T4 DNA ligase and its 10X buffer.
(11) JMJ09 receptor bacteria.
(12) Falcon 2059 polypropylene tubes.
(13) SOC medium.
(14) LB plate medium (100 μg/ml ampicillin).
5. Clone screening
(1) TE buffer: 10 mmol/L Tris- HCl ( pH 7.4), 0.1 mmol/L EDTA.
(2) PCR reaction system, see 3 "RNA-rPCR method".
(3) SP6 and T7 promoter primers.
(4) Phenol: chloroform (1 : 1), keep away from light.
(5) Chloroform: isoamyl alcohol (24 : 1).
(6) 3 mol/L sodium acetate (pH 5.2).
(7) Non-denaturing agarose gel.
(8) Electrophoresis buffer 1X TBE.
(9) 10X non-denaturing agarose gel top-up buffer, see 3 "RNA-rPCR Methods".
(10) fmol DNA Cycle Sequencing System (Promega).
6 Instrumentation
(1) Miniature gel horizontal electrophoresis apparatus.
(2) Ultraviolet transilluminator.
(3) PCR amplifier.
(4) High-speed cryogenic centrifuge.
(5) Cryogenic microcentrifuge.
(6) Benchtop cryogenic centrifuges.
(7) Rotary vacuum concentrator (Speed-Vac concentrator).
II. Experimental Methods
1. Preparation of cell extracts
(1) Cultivate 3~4 bottles of cells in 150 cm2 tissue culture flasks.
(2) Wash three times with 10~15 ml ice-cold PBS. Collect the cells with a cell scraper, resuspend the cells with ice-cold PBS and transfer them to a 50 ml sterile centrifuge tube.
(3) Centrifuge at 1000 g at 4℃ for 10 min.
(4) Resuspend the cells with 0.5 ml of freshly prepared NET-2 buffer containing 100 U RNase inhibitor.
(5) The cell resuspension was ultrasonicated on ice for 5~10 s three times.
(6) Transfer the ultrasonicated cell suspension to a 15 ml pressure-resistant permeable glass tube and centrifuge at 10,000 g for 10 min at 4°C. Recover the supernatant and use it for the immunoprecipitation reaction. The supernatant can be frozen at -80°C, but it is preferable to carry out subsequent operations immediately.
2. Immunoprecipitation
(1) To remove non-specific binding of protein agarose gel from whole cell extracts, add an equal volume of protein A agarose gel to a cell extract with 1 to 1.5 mg of total cellular protein and mix by rotation at 4°C for 30 min.
(2) Centrifuge at 10000 g for 2 min at 4°C to remove the protein A agarose gel and recover the supernatant for use.
(3) Add specific antibody, 40 μl of 1 mg/ml yeast tRNA and 30 μl of RNase inhibitor to the treated cell extract and incubate at 4°C for 1 h. Gently turn the mixture during incubation.
(4) Add 2 times the volume of protein A agarose gel to the cell extract, mix gently and incubate at 4℃ for 30 min.
(5) Centrifuge at 10000 g for 2~3 min at 4℃, and wash the precipitate with 350 μl NET-2 buffer at least 4~5 times.
(6) Add 350 μl NET-2 buffer and phenol: chloroform (1:1), and vortex vigorously for 1 min.
(7) Centrifuge at 10000 g at 4℃ for 5 min.
(8) Add 10 U RQI DNAase to the aqueous phase and incubate at 37°C for 15 min to remove contaminating DNA.
(9) Extract with equal volume of phenol: chloroform (1:1).
(10) Centrifuge at 10000 g at 4°C for 5 min.
(11) Extract the aqueous phase again with an equal volume of chloroform: isoamyl intoxicant (24 : 1 ).
(12) Centrifuge at 1000 g at 4°C for 5 min.
(13) Precipitate RNA with 0.1 v/v of 3 mol/L sodium acetate, 2.5 v/v of ethanol and 20 μg of glycogen, which helps to precipitate trace amounts of nucleic acids. Samples are placed in a dry ice/ethanol bath for at least 30 min.
(14) Centrifuge at 10,000 g at 4°C for 30 min.
(15) Discard the supernatant and wash the precipitate with 80% ethanol.
(16) Centrifuge at 10000 g at 4°C for 5 min.
(17) Concentrate and dry in a rotary vacuum concentrator, and dissolve the precipitate in 15-20 μl of water.
3. RNA-rPCR method
(1) Take half of the RNA sample prepared by immunoprecipitation, heat it at 65 ℃ for 5 min, and then put it on ice to cool quickly.
(2) Reverse transcription was performed in a total volume of 50 μl with the following reaction system: 50 mmol/L Tris-HCl (pH 8.3), 10 mmol/L DTT, 3 mmol/L MgCl2, dNTP (500 μmol/L each), 1 U RNase inhibitor, 50 U MMLV reverse transcriptase, 150 ng of Universal Primer dN6 ( 5'-GCCGCTCGAGTGCAGAATTC- NNNNNNN-3').
(3) The reaction was incubated at 42 ℃ for 1 h. At the end of the reaction, the reaction was heated at 95 ℃ for 3 min, and then quickly cooled on ice.
(4) Add 1 μl of RNase H and incubate at 37 ℃ for 15 min to remove the RNA strand.
(5) Heat the sample at 95 ℃ for 3 min, and cool on ice.
(6) Synthesize the second strand of cDNA, add 100 ng of universal primer-dN6 (5'-GC-GC-CGCTCGAGTGCAGAATTCNNNNNNNN-3') to 41.5 μl of the first strand, heat at 95℃ for 3 min, and then cool on ice quickly. Add 5 μl of 10X Klenow reaction buffer, 1 μl of dNTPs (25 mmol/L each), 0.5 μl of 100 mmol/L DTT, and 1 μl (6U) of Klenow enzyme to a final volume of 50 μl. Incubate at 37°C for 30 min.
(7) The samples were extracted with phenol:chloroform (1:1) and chloroform:isoamyl alcohol (24:1). The samples were purified by centrifugation on an RNase-free G-50, D-RF column to remove the excess of the universal primer, dN6.
(8) Take 1/10 volume of double-stranded cDNA as PCR amplification template, then add 10X PCR buffer, 2.5 μl dNTPs (10 mmol/L each), 100~200 ng universal primer (5'-GCCGCTCGAGTGCAGAATTC-3') and DEPC treated water to a total volume of 50 μl, and then 1.5 μl Taq DNA polymerase. l Taq DNA polymerase was added. The amplification conditions were as follows: 94°C for 1 min, 55°C for 1 min, 72°C for 3 min, and a total of 40 cycles were amplified.
(9) In order to ensure the integrity of the synthesized double-stranded cDNA, an additional PCR reaction was performed after the completion of PCR. Add 10X PCR buffer, 6 μl of dNTP, 100 ng of universal primer, 3 μl of Taq enzyme to 30 μl of PCR reaction solution, and then add appropriate amount of DEPC treated water to a final volume of 300 μl.
(10) Mix gently and amplify for 3 cycles at 94℃ for 30 seconds, 50℃ for 1 minute and 72℃ for 2 minutes. Then the reaction was carried out for 4 cycles at 50°C for 1 min and 72°C for 5 min.
(11) The PCR products were extracted with an equal volume of phenol: chloroform (1:1), precipitated with 30 μl of 3 mol/L sodium acetate and 750 μl of ethanol, and resuspended in 20-30 μl of DEPC water.
(12) One-tenth of the PCR product was analyzed by 1% nondenaturing agarose gel electrophoresis.
4. Construction of cDNA plasmid libraries and transformation
(1) The cDNA was recovered using a PCR product recovery kit and the remaining primers were removed.
(2) The purified amplified cDNA product was digested with 100 U of EcoRⅠ in a final volume of 150 μl and reacted at 37 °C for 2 h. The reaction was carried out at 37 °C for 2 h.
(3) Simultaneously digest 500 ng of plasmid pGEM-7Z with 10 U of EcoRⅠ in a final volume of 10 μl at 37°C for 2 h. Add 2 μl of calf intestinal alkaline phosphatase (CIAP), 10 μl of 10X CIAP Reaction Buffer, and DEPC-treated water to a final volume of 100 μl and incubate at 37°C for 30 min. Add 2 μl 0.5 mol/L EDTA at 65°C, and then incubate for 30 min. The reaction was terminated by adding 2 μl of 0.5 mol/L EDTA and holding at 65 ℃ for 20 min.
(4) Extract the two EcoR Ⅰ digestion products with equal volume of phenol: chloroform (1:1).
(5) Centrifuge at 10000 g for 5 min at 4 ℃.
(6) The aqueous phase was extracted with an equal volume of chloroform: isoamyl alcohol (24:1).
(7) Centrifuge at 10000 g for 5 min at 4 °C.
(8) Add 0.1 volume of sodium acetate and 2.5 volume of ethanol to the supernatant and leave for 30 min in a dry ice/ethanol bath to precipitate the DNA.
(9) Centrifuge at 10000 g at 4°C for 30 min.
(10) Wash the precipitate with 1 ml of 80% ethanol.
(11) Centrifuge at 10000 g at 4°C for 5 min.
(12) Dry the sample with a rotary vacuum concentrator and add 10-12 μl of DEPC-treated water to dissolve the precipitate.
(13) Ligate 1-5 ng of EcoR Ⅰ-treated amplified cDNA with 100 ng of EcoR Ⅰ-treated and dephosphorylated pGEM-7Z vector using T4 DNA ligase, add 1.5 μl of T4 DNA ligase and 1.5 μl of 10X buffer, and then add a final volume of 15 μl.
(14) Ligation was performed at room temperature for 3~3.5 h. The samples could be stored at 4℃ before transformation.
(15) Take 5 μl of ligation product and add it to 100 μl of JM109 sensory bacteria, mix gently and ice bath for 30 min.
(16) Heat shock at 42℃ for 2 min.
(17) Quickly place on ice for 2 min.
(18) Add 1 ml of SOC Liquid Medium preheated to 37 °C.
(19) Incubate at 37°C for 1 h with shaking at 150~200 r/min.
(20) Take 100 μl and spread it evenly on LB/Aminobenzyl plate medium.
(21) Incubate at 37℃ overnight.
5. Clone Screening
(1) Use PCR to identify the clones of the inserted fragments. Dip a toothpick into each clone and add it to 100 μl TE.
(2) Heat at 100°C for 10 min. store the template at -20°C for use.
(3) Mix 10 μl of template, 10 μl of 10X PCR buffer, 8 μl of dNTP (10 mmol/L each), 100 ng of SP6 promoter primer, 100 ng of T7 promoter primer, 1 μl of Taq enzyme, and replenish with sterile water to a final volume of 100 μl.
(4) PCR amplification was performed at 94°C for 40 s, 50°C for 1 min, and 72°C for 2 min for a total of 35 cycles.
(5) The PCR products were analyzed on a 1% nondenaturing agarose gel.
(6) Sequence analysis of positive clones.