Conjugate transcription profiling experiments
Conjugate transcription profiling experiments
Nuclear contig analysis is used to determine whether a gene is transcriptionally regulated in a given clone. Theoretically, nuclear contig analysis should be performed after hybridization experiments that detect changes in steady state mRNA levels and before promoter analysis. This experiment was derived from the next volume of the Molecular Cloning Laboratory Guide (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Conjugate transcription profiling experiments
Principle
Centrifuge and rotary head, boiling water bath, Dounce homogenizer, crystalline plastic or ultra-bright boom tubes, polypropylene tubes, blades, oscillating water bath preset to 30°C, water baths preset to 4°C and 65°C
Materials and Instruments
Non-recombinant plasmid vector Recombinant plasmid containing cDNA or gene of interest Cultured cells or fresh tissue Move makings For more product details, please visit Aladdin Scientific website.
Chloroform DNA denaturing solution Ethanol Glycerol Storage buffer HSB buffer Labeling buffer LiCl Lysis buffer NaCl NaOH Nonidet P-40 Nucleus washing buffer Phenol phosphate buffer Pre-hybridization solution Hybridization solution Reaction buffer RNasin SDS Termination buffer Tissue homogenization buffer Taipan blue dye DNA enzyme solution Proteinase K Restriction enzymes Radioactive compounds
Centrifuges and rotary heads Boiling water baths Dounce homogenizers Crystalline plastic or ultra-bright boom tubes Polypropylene tubes Blades Oscillating water baths preset to 30°C Water baths preset to 4°C and 65°C
Important: All tubes and solutions must be free of RNAase.
Buffers and solutions
See Appendix 1 for components of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
Chloroform
Chloroform: isoamyl alcohol
DNA denaturing solution
2mol/L NaCl
0.lmol/L NaOH
Ethanol
Glycerol storage buffer
50 mmol/L Tris-Cl (pH 8.3)
5 mmol/L MgCl2
0.1 mmol/L EDTA(pH8.0)
40% (V/V) glycerol
Buffer stored at 4°C
HSB Buffer
10 mmol/L Tris-Cl (pH 7.4)
50 mmol/L MgCl2
2 mmol/L CaCl2
0.5mol/L NaCl
buffer was stored at room temperature.
Labeling buffer
20 mmol/L Tris-Cl (pH 8.0 at 4°C)
140 mmol/L KCl
10 mmol/L MgCl2
1 mmol/L MoCl2
20% (V/V>glycerol)
14 mmol/L β-liucoethanol
1 mmol/L each ATP, GTP and CTP
10 mmol/L phosphocreatine
100ug/ml phosphoinositide kinase
0.1 umol/L [32P ]UTP, 500~5000 uCi/ml
The last 5 components are added just before use and the solution is stored at 4°C. The radioactive UTP requires special protection after addition.
LiCl(5mol/L)
Lysis Buffer
10 mmol/L Tris-Cl (pH 8.4 at 4°C)
1.5 mmol/L MgCl2
0.14 mol/L NaCl
buffer stored at 4°C.
NaCl (2mol/L)
NaOH(0.1mol/L)
Nonidet P-40(5% V/V)
Cell nucleus washing buffer
20 mmol/L Tris-Cl (pH 8.0 at 4°C)
140 mmol/L KCl
10 mmol/L MgCl2
1 mmol/L MoCl2
20%(v/v) glycerol
14 mmol/L β-mercaptoethanol
Store the buffer at 4°C. Add the β-mercaptoethanol concentrate to the buffer before use.
Phenol
Phosphate Buffer Solution (PBS)
Pre-Hybridization Solution/Hybridization Solution
Hybridization buffers containing formamide, such as 50% (V/V) formamide, 6XSSC, 5 mmol/L sodium pyrophosphate, 2XDenhardt's solution, 0.5% (m/V) SDS, 10ug/ml poly(A), and 100ug/ml salmon sperm DNA, are generally used in nucleo-linkage experiments.
2X reaction buffer
10 mmol/L Tris-Cl (pH 8.0)
5 mmol/L MgCl2
0.3 ml/L KCl
Store the buffer at 4°C. Add 5ul of 1 mol/L DTT, 100ul of 100 mmol/L ATP, 10ul of 100 mmol/L CTP, and 10ul of 100 mmol/L GTP to 1 ml of 2x reaction buffer before use.
RNasin
RNasin is the common name for the rat liver RNAase inhibitor protein. Some companies offer this enzyme product.
SDS (0.5%m/V)
6XSSC
Termination buffer
50 mmol/L Tris-Cl (pH 7.5)
20 mmol/L EDTA (pH 8.0)
0.8% (m/V) SDS
buffer was stored at room temperature.
Tissue homogenization buffer
10 mmol/L HEPES-KOH (pH 7.6)
25 mmol/L KCl
0.15 mmol/L arginine
0.5 mmol/L spermidine
1 mmol/LEDTA(pH8.0)
2 mol/L sucrose
10% (V/V) glycerol
Store the buffer at 4°C. Prior to use in Step 1, add CTT to a final concentration of 1 mmol/L and protease inhibitors (0.5 mmol/L phenylmethylsulfonyl fluoride, 1ug/ml leucinopeptide, 1ug/ml pepstatin, or other reagents as required).
Taipan blue dye (0.4% m/V)
Dissolve a certain amount of the dye in Ca2+ and Mg2+ free phosphate buffer solution (PBS). Store at room temperature.
Enzymes and buffers
DNA enzyme solution
Dissolve 2 mg of RNAase-free DNA enzyme (e.g., Worthington) in 1 ml of 0.0025 mol/L HCl and 50% (V/V) glycerol. Store the solution at -20°C.
Protease K (optional)
Restriction enzymes
See step 9.
Radioactive compounds
[ α-32P ]UTP (500~5000uCi/ml)
Each nucleic acid sample was radiolabeled with 100uCi.
Centrifuge and rotor head
Beckman SW28 turntable or equivalent
Sorvall H1000B Turn Head or equivalent
Sorvall SS-34 turn head or similar.
IMPORTANT: All heads are pre-cooled to 4°C.
Specialty Equipment
Boiling water bath
Dounce homogenizer with type B mortar and pestle
Crystal Plastic or Ultra-Bright Bucket Centrifuge Tubes
Polypropylene Tubes (17 mmx100 mm)
Blades
Oscillating water bath preset to 30°C
Preset to 4°C and 65°C water baths
Auxiliary reagents
Step 11 of this program requires the reagents and equipment listed in Chapter 7, Program 9.
Step 13 of this protocol requires the reagents listed in Chapter 6, Option 8.
Steps 14 to 16 of this protocol require the reagents listed in Chapter 6, Options 8 and 10.
Vectors and Strains
Non-recombinant plasmid vectors
Recombinant plasmids containing the cDNA or gene of interest
Cells and Tissues
Cultured cells or fresh tissue
Methods
Nucleic acid isolation and radiolabeling of RNA transcripts
1. Isolation of nucleic acids from cultured cells or fresh tissue.
Isolation of nucleic acids from cultured cells
a. Scrape the cells from the petri dish and wash them twice with ice-cold PBS. In a polypropylene tube (17 mmXl00 mm), resuspend 1x107 to 1X108 ruffles with 1 ml ice-cold lysis buffer. Add 2~4ul 5% NonidetP-40, and ice bath the suspension for 10 min.
b. Mix 10ul of suspension with an equal volume of 0.4% Taipan blue dye and examine the solution under a microscope equipped with a 20x objective. Lysed cells absorb the dye and appear blue, while non-lysed cells remain transparent because the dye is not permeable. Continue to add 2ul of 5% NonidetP-40 and observe cell lysis until >80% of the cells are lysed.
c. Recover nucleic acids by centrifugation in a benchtop centrifuge at 1300 g (Sorvall Hl000B head 2500r/min) for 1 min. Remove the supernatant. The nucleic acid precipitate is washed twice with lml Nucleic Acid Wash Buffer. Proceed to step 2.
Isolation of Clostridial Acid from Tissue
a. Dissect 10-15 g of tissue and mince. Adjust volume of chopped tissue to 30 ml with ice-cold Tissue Homogenization Buffer and homogenize in a tight-fitting Dounce homogenizer.
b. To detect lysis, 10 ul of cell suspension is mixed with an equal volume of 0.4% Taipan blue dye and the solution is examined under a microscope equipped with a 20X objective. Lysed cells absorb the dye and appear blue in color, while non-lysed cells remain transparent because the dye is not permeable. The homogenization is continued until >80%~90% of the cells have ruptured.
c. Dilute homogenate with ice-cold Tissue Homogenization Buffer to 85 ml. Spread 27 ml of homogenate on a 10 ml cushion of ice-cold Homogenization Buffer in a crystalline plastic or ultra-bright bucket centrifuge tube. Centrifuge the tubes at 4°C for 40 min at 103,900g (Beckman SW28 turntable 24000r/min).
d. Remove supernatant and invert tubes for 1 to 2 min. Place tubes on ice. Resuspend the precipitate by blowing up and down with 2 ml of glycerol storage buffer.
(Optional) Cut off the top 2/3 of the tube with a razor blade and place the bottom 1/3 of the tube containing nucleic acids on ice before resuspending the precipitate in glycerol storage buffer.
e. 10ul of nucleic acid suspension is mixed with 990ul of 0.5% SDS. Measure OD260 with a UV spectrophotometer and dilute the nucleic acid suspension with glycerol storage buffer to a final concentration of 50 OD260/ml. Dispense the nucleic acid preparation into 200ul portions in 1.5 ml centrifuge tubes, freeze in liquid nitrogen, and store at -70°C. Proceed to step 2.
2. Radiolabel the nascent RNA in the purified nucleic acid.
For nucleic acids isolated from cultured cells, radiolabel the transcripts.
a. After the last wash (step 1c), remove as much supernatant as possible and resuspend the nucleic acids in 50-100ul of labeling buffer. Incubate the nucleic acids in a 30°C oscillating water bath for 15-20 min.
Warning: When radioactive UTP is added to the solution, be extremely careful to wear gloves and glasses and not to contaminate the benchtop with highly radioactive solutions.
b. Centrifuge the nucleic acids in a bench-top centrifuge at 800 g (Sorvall H1000B head 1960r/min) for 5 min. Precipitate the nucleic acids, carefully remove the supernatant, and dispose of as radioactive waste. Proceed to step 3.
Addition of [32P ]UTP to RNA can be detected by trichloroacetic acid (TCA) precipitation as described in Appendix 8. For a successful labeling reaction containing radiolabeled UTP and 10OD260 nucleic acid, 80% to 90% of the 32P will be incorporated into material that can be precipitated with TCA.
For nucleic acids isolated from tissues, radiolabeling the transcripts therein
a. Transfer an appropriate number of cell nuclei preparations from -70°C to an ice bucket. After the liquid has dissolved, add 400 units of RNasin per tube. 200ul of 2X Reaction Buffer supplemented with nucleotides and DTT is added to each tube of nucleic acids. Add 100 uCi [ α-32P ]UTP.
WARNING: Use extreme caution after radioactive UTP has been added to the solution. Wear gloves and glasses and take care not to contaminate the benchtop with highly radioactive solutions, etc.
b. Incubate the nucleic acids in a shaking water bath at 30°C for 20 min. Centrifuge at 2000 r/min for 1-2 min to recover the nucleic acids. Carefully remove the supernatant and dispose of as radioactive waste. Proceed to step 3.
Addition of [32P ]UTP to RNA can be detected by trichloroacetic acid (TCA) precipitation as described in Appendix 8. For a successful labeling reaction with 100uCi radiolabeled UTP and 10OD260 nucleic acid, 80% to 90% of the 32P will be incorporated into material that can be precipitated with TCA.
3. Resuspend the nuclear precipitate with 1 ml of ice-cold HSB buffer and add 10 ul of DNAzyme solution. Blow the nucleic acid up and down until the nucleic acid is resuspended and the viscosity of the solution decreases (1-5 min). Add 2 ml of termination buffer.
The DNAzyme treatment reduces the viscosity of the solution and prevents nascent radiolabeled RNA transcripts from being captured by genomic DNA. The viscosity is not completely eliminated, and nucleic acid clumps are often left behind.
4.(Optional) To increase the yield of radiolabeled RNA, after the DNAzyme treatment, proteinase K can be added. After adding 2 ml of termination buffer, add proteinase K to a final concentration of 100 ug/ml. Incubate the solution at 42°C for 30 min. proceed to step 5.
For a larger scale protocol for DNA removal and radiolabeled RNA purification, see Groudine et al (1981).
5. Add 3 ml phenol and incubate the mixture at 65°C for 15 min, shaking every 5 min during this period. Add 3 ml chloroform/isoamyl alcohol, shake, and separate the organic and aqueous phases by centrifugation at 1900 g (Sorvall Hl000B head 3000 r/min).
The aqueous phase was again extracted with 3 ml of chloroform, centrifuged as before and transferred to a new tube.
6. Add 0.3 ml 5mol/L LiCl and 2.5 times the volume of ethanol and mix well. Collect the nucleic acid precipitate by centrifugation at 12000 g (Sorvall SS-34 turntable 10000r/min) for 10 min.
7. Resuspend the precipitate with 0.4 ml H2O and transfer to a 1.5 ml centrifuge tube. Add 40ul of 5mol/L LiCl and 2.5 times the volume of ethanol. Centrifuge the solution at maximum speed for 10 min.
8. Resuspend the precipitate with 100ul H20 and measure cpm/ul in a liquid flash counter.
Hybridization of RNA to immobilized DNA
9. 10ug of recombinant plasmid DNA containing the cDNA or gene of interest and 10ug of empty plasmid vector are linearized with restriction enzymes using restriction enzyme cleavage sites that must be present in the vector sequence.
10. The cut DNA is recovered by standard ethanol precipitation and the precipitates are resuspended separately in 20ul of DNA denaturing solution. the DNA resuspension is boiled for 2 min, then 180ul of 6XSSC is added to each tube.
11. Cut a piece of nylon or nitrocellulose membrane of a size suitable for dot or slit hybridization apparatus. Wet the membrane with water and soak it in 6XSSC for 5-10 min. Clamp the wet membrane to the hybridization apparatus, connect a vacuum tube and a pump (see Scheme 9 in Chapter 7).
12. Denatured DNA is filtered through each of the separated slits and each slit is washed with 200ul of 6xSSC.
13. Remove the hybridization apparatus, allow the membrane to air dry, and then fix the DNA to the membrane by baking or UV exposure.
14. Incubate the membrane in the prehybridization solution for at least 16 h at a suitable temperature (e.g., 42°C for solvents containing 50% formamide).
15. The radiolabeled probe (32P-labeled RNA from step 8. 2x106~4X106cpm/ml ) is added directly to the prehybridization solution, and the membrane is filtered and reincubated for 72 h. The membrane is then incubated for at least 16 h at the appropriate temperature (e.g., 42°C for solvents containing 50% formamide).
16. Wash the membrane under high rigor conditions and expose the X-ray film or phosphor plate. The exposure time is usually 24-72 h for X-ray film and 4-24 h for phosphor plate.