nuclear transcription analysis
Nuclear runoff (runoff) analysis can be used to examine the transcriptional efficiency of specific genes in isolated cell nuclei. For runoff analysis, the nucleus is isolated in vitro and transcribed in the presence of radioisotope-labeled nucleotides. The synthesized KNA is labeled with the isotope, and the hybridization of this mixed RNA with a DNA probe immobilized on a nitrocellulose filter membrane h for a particular gene reflects the transcriptional state and rate of transcription of that gene.
Operation method
nuclear transcription analysis
Materials and Instruments
cDNA 0.45um cellulose nitrate Nylon membrane Yeast tRNA Move I Materials and equipment Caveat 1) The time for washing the membrane can be determined on an experimental basis. Consider the nature of the probes used and strive to minimize background.2) The quality of the nuclei is critical for nuclear uncontrolled transcription assays. the Dounce homogenizer helps to prepare nuclei from cells that are less susceptible to NP40 cleavage. For more product details, please visit Aladdin Scientific website.
PBS NP-40 Lysis Buffer: 20XSSC LNaOH Glycerol Storage Buffer 10X Transcription Buffer Nucleotides: ATP GTP CTP 200uCi "α32P]-UTP 10XSET Protease K DNase without RNase CaCI2 Guanidine thiocyanate solution Tris-SDS buffer Water-saturated phenol Chloroform Isopentanol Ethanol anhydrous Isopropyl Alcohol Sodium Acetate
UV Crosslinker D Slit Blotter Hybridization Chamber
1) cDNA
2)PBS
3)NP-40 lysis buffer: 0.5% NP-40, 10 mmol/LTris-HCl(pH7.4), 3 mmol/LMgCl2, 10 mmol/LNaCl.
4)20XSSC:3mol/LNaCl,0.3mol/L sodium citrate (pH7).
5)1mol/LNaOH
6) Glycerol storage buffer: 40% glycerol, 50 mmol/L Tris-HCl (pH8.3) 0.5 mmol/L MgCl2, 0.lmmol/L EDTA
7) 10X Transcription buffer: l00 mmol/L Tris-HCl (pH7.5), 50 mmoI/L MgCl2, 800 mmol/L KCllmmol/L EDTA, 5 mmol/L DTT
8) Nucleotides:ATP,GTP,CTP
9) 200uCi " α32P ]-UTP:3000Ci/nmol.
10) 10XSET;5% SDS, 50 mmol/LEDTA, 50 mmol/LTris-HCl(pH7,4)
11) Protease K
12) DNase without RNase: 1Omg/mL
13) CaCI2: 20 mmol/L
14) Guanidine thiocyanate solution: 4 mol/L guanidine thiocyanate, 25 mmol/h sodium citrate (PH7.0), 0.5% Sa rcosyl0.lmol/L2-hydrophobic ethanol
15) Tris-SDS buffer: Tris-HCl (PH7.2), 1 mmol/LEDTA, 0.1% SDS.
16) Water-saturated phenol
17) Chloroform: isoamyl alcohol (49:1)
18) 0.45um cellulose nitrate/nylon membrane.
19) Anhydrous ethanol.
20) Isopropyl alcohol.
21) Yeast tRNA: 10 mg/ml.
22) Sodium acetate:2mol/L
23) UV crosslinker D
24) Slit blotting device
25) Hybridization box.
II. Methods of operation
Each of the following steps was performed at 0-4°C
1) Wash 1X107-5X107 cells with cold PBS and centrifuge at 1500 g for 5 min to collect the cells.
2) Remove the supernatant, resuspend the cells in PBS and centrifuge at 1500 g for 5 min to collect the cells.
3) Remove supernatant, add 4 ml of NP-40 lysate, gentle vortexing, and place on ice for 10 min.
4) Centrifuge the cells at 400 g for 10 min at 4℃ to precipitate the nuclei. Wash once with NP-40 lysate.
5) Resuspend the precipitate with 200ul of glycerol buffer and observe the cell lysis under microscope.
6) Dispense and freeze in liquid nitrogen.
(ii) Nuclear transcription
1) In a sterile centrifuge tube, add 3X107 nuclei, 35% glycerol, 20ul1OX transcription buffer, 4 mmol/L ATP, GTP, CTP, 200uCi( α32P ]-UTP, 200ul of distilled water, incubate at 26°C for 10 min.
2) Add 10ul_DNase1, 10ul20 mmol/LCaCl2, incubate at 26°C for lOmin, and digest nuclear DNA.
3) Add 2ul Protease K, 25ul10×SET, 5ul yeast tRNA, incubate at 37℃ for 30 min.
4) Add 550ul thiocyanoguanidine solution, 90ul2mol/LNaAc, 900ull water saturated phenol, 180ul chloroform/isoamyl alcohol, placed on ice for 15 min.
5) Centrifuge at 4°C, 12000 g for 15 mm and aspirate the aqueous phase.
6) Add an equal volume of isopropanol and precipitate for 1-2 h at 20 ℃.
7) Centrifuge at 12000 g at 4℃ for 15mim and dissolve the precipitate with 300ul of guanidine thiocyanate solution.
8) Add an equal volume of isopropanol and precipitate at 20°C for 1-2 h, and the precipitate is dissolved in 300ul of guanidine thiocyanate solution.
9) Centrifuge at 12000 g for 15mim at 4℃, wash the precipitate with 70% ethanol and dissolve the precipitate with 1 ml of Tris-SDS buffer. The total activity was 1X107-2X107cpm.
(iii) Hybridization analysis
1) Add 10% lmol/L NaOH to 100ul of 25ug cDNA probe or 50ug linear plasmid to denature it.
2) Incubate for 15 min, neutralize with 10x volume of 6XSSC.
3) Cut a nitrocellulose/nylon membrane of appropriate size. Soak the membrane in 0.4 mol/L Tris (PH7.0) solution for 30 min, and then place the membrane on the slit blotting device.
4) Add 1-5ug of DNA onto the membrane and bake at 80℃ for 2 h under vacuum or crosslink for 2 min under UV.
5) Pre-hybridization and hybridization were performed according to conventional methods.
6) X-ray film, develop and analyze.