Differential cloning is an effective technique for isolating and characterizing mRNAs that are differentially expressed in two cell populations. The cell types to be compared are [+] (or test) cells and [-] (or driver) cells, and mRNAs that are expressed in the test cells but not in the driver cells will be isolated. The number of necessary subtractions depends mainly on the diversity of the cDNAs. Diversity is the total number of different cDNA or cDNA fragments per cell type (Davidson, 1986). Source: Comprehensive Molecular Biology Laboratory Guide, Fifth Edition.
Operation method
basic program
Materials and Instruments
Double-stranded cDNA (ds cDNA) for class A and B cells Move For materials, reagents, and consumables, see "Others". 1. Prepare 2 restriction endonuclease digestion reactions (AluI and AluI+Rsal) for each set of ds cDNA (A and B): 30 ng ds cDNA 3 ul 10× AluI buffer 10 U AluI or 10 U AluI + 10 U RsaI Add water to 30.0 ul. Incubate overnight at 37°C to ensure complete digestion. It is best to use enzymes that produce flat ends. If the enzyme does not produce flat ends, additional flattening or flattening is required. 2. Inactivate the endonuclease by incubating at 65°C for more than 10 min. 3. 3. For kinase treatment of oligonucleotides a1 and b1, use the following reaction systems (25 ul each): 18.0 ul H2O 2.5 ul 10 mmol/L ATP 2.5 ul 10×T4 Polynucleotide Kinase Buffer 1.5 ul 3 ug/ul of oligonucleotide a1 or b1 0.5 ul 10 U/ul T4 polynucleotide kinase buffer incubate at 37℃ for 60 min. 4. 4. Inactivate the kinase by incubation at 65°C for 20 min. 5. 5. Add 1.5 ul of oligonucleotide a2 or oligonucleotide b2 to form a1/a2 or b1/b2 linker. Mix well and centrifuge at maximum speed. incubate at 45°C for 10 min. 6. 6. In a 0.5 ml PCR tube, prepare a ligation reaction (130 ul per reaction) for each cDNA set using the appropriate linker: 63 ul H2O 13 ul 10× T4 DNA Ligase Buffer 30 ul 40% PEG 8000 1 ul 15 mmol/L ATP 10 ul AluI digested cDNA 10 ul AluI/RsaI digested cDNA 2 ul a1/a2 or b1/b2 ligand 1 ul 10 U/ul T4 DNA ligase Mix well and incubate at 16°C for 2 h. 7. Place the reaction tube on ice. 7. Place the reaction tube on ice for more than 10 min. 8. Prepare a Sephacryl S-300 centrifuge column according to the manufacturer's instructions. 9. 9. Add 1 ul 75 mmol/L ATP and 1 ul T4 polynucleotide kinase to each ligation reaction and incubate at 37°C for 30 min. 10. 10. The ligations were sequentially extracted with 1x volumes of 25:24 phenol/chloroform and chloroform. 11. 11. Centrifuge the ligated products on a Sephacryl S-300 column to remove the unconjugated linker, and then centrifuge at 400 g for 2 min at room temperature using a bucket rotor on a Beckman Accuspin FR centrifuge. 12. 12. For two sets of cDNA, each set was prepared with 50 ul of PCR reaction system. 35 ul of H2O was added to the reaction system: 35 ul H2O 5 ul 10×Taq DNA Polymerase Buffer 3 ul 25 mmol/L MgCl2 1 ul 10 mmol/L 4dNTP mixture 0.5 ul 2.5 ug/ul oligonucleotide a2 or oligonucleotide b2 5 ul 0.2 ng/ul ligated cDNA A or cDNA B 0.5 ul 5 U/ul Taq DNA polymerase Add a few drops of sterile PCR-grade mineral oil to cover the reaction system. 13. Amplify the cDNA using the following PCR program: 30 cycles: 1 min 94°C (denaturation) 1 min 50°C (denaturation) 2 min 72°C (extension) 25 s 72°C (auto-extension) For thermal cyclers without automatic extension, the extension time is 2-4 min. 14. 14. analyze 5-10 ul of amplified cDNA by agarose gel electrophoresis to determine the size range of amplified cDNA (150 bp-1.5 kb, mostly 250 bp). 15. analyze two sets of amplified cDNA by agarose gel electrophoresis. 15. For each of the two sets of amplified cDNAs, prepare the following PCR reaction systems (100 ul each) for the radiolabeled test cDNAs: 77 ul H2O 10 ul 10×Taq DNA polymerase buffer 6 ul 25 mmol/L MgCl2 2 ul 10 mmol/L 4dNTP mixture 1 ul Diluted [ 32P ] dCTP 1 ul 2.5 ug/ul oligonucleotide a2 or oligonucleotide b2 2 ul A0 or B0 cDNA (~0.4 ug) 1 ul 5 U/ul Taq DNA polymerase Add a few drops of sterile PCR-grade mineral oil to cover the reaction system. 16. 16. For each of the two sets of amplified cDNAs, prepare 3 or 4 PCR reactions (100 ul each) with biotin-labeled driver cDNA: 73.3 ul H2O 10 ul 10×Taq DNA polymerase buffer 6 ul 25 mmol/L MgCl2 6.7 ul drive dNTP 1 ul 2.5 ug/ul oligonucleotide a2 or oligonucleotide b2 2 ul cDNA A0 or cDNA B0 (1 to 5 ng) 1 ul 5 U/ul Taq DNA polymerase Add a few drops of sterile PCR-grade mineral oil to cover the reaction system. 17. 17. Test and drive cDNA synthesis using the PCR amplification program in Step 13. 18. Purify the amplified cDNA with a commercial anion-exchange PCR centrifuge column (Qiagen) according to the manufacturer's instructions, removing unincorporated nucleotides, primers, and salts. 19. Quantify the product nucleic acids by spectrophotometry. 20. Prepare 2 hybridization reactions ([ 32P ] An-Bio-Bn and [ 32P ] Bn-Bio-An). Precipitate 1 ug of radiolabeled test DNA and 20 ug of biotin-labeled driver DNA with ethanol in a 1.5 ml silanized microcentrifuge tube; do not freeze. Air-dry the precipitate, and when it is just dry, gently blow on it with a pipette tip and redissolve it in 5 ul HEPES buffer. Detect the re-dissolved nucleic acid solution with a handheld Geiger counter. 21. Transfer the re-dissolved DNA into a 0.5 ml PCR tube. Add 5 ul of 2× Hybridization Buffer at 68°C for subtractive hybridization. Blow gently to mix and add a few drops of sterile PCR-grade mineral oil to cover the DNA solution. Centrifuge at maximum speed for a few seconds. 22. Heat both tubes at 95°C for 10 min, then slowly cool to 68°C within 1 h and continue to incubate at 68°C for 2 h (fast hybridization). 23. Mix 7 ul 1 mol/L NaCl and 140 ul HEPES buffer and heat to 68°C. Add to the hybridization reaction to dilute. Add to the hybridization reaction to dilute the reaction. Mix well and centrifuge briefly at maximum speed. Cool to room temperature. 24. Remove 5 ul from each tube and save (use as a total amount of phenol extraction prior to calculation). 25. Add 15 ul of Streptavidin to each tube, vortex and incubate for 5 min at room temperature. 26. Extract each tube with an equal volume of 25:24 phenol/chloroform. Retain the aqueous phase and transfer to a new tube. 27. Add 10 ul of streptavidin to the aqueous phase of each tube. Mix well and incubate at room temperature for 5 min. 28. 28. Extract twice with phenol/chloroform and twice with chloroform. Measure the volume of the aqueous phase in each tube. 29. Remove 5 ul from each tube and store (to be used as a total after phenol extraction). The percentage of test cDNA removed was estimated using the following equation: % Test cDNA removed = 100 i (total amount after phenol extraction × 100/total amount before brew extraction) 30. Repeat the process of differential subtraction with An or Bn test cDNA and a suitable driver cDNA (steps 15 to 29). The selection of the driver cDNA is determined by the strategy of differential reduction. The An or Bn driver cDNA is used for fast hybridization (2 h) and the A0 or B0 driver cDNA is used for long hybridization (30-40 h). The process of differential subtraction was detected by gap spot hybridization. 31. Amplify 5 ul of subtracted cDNA (An and Bn) using the procedure in step 13. Purify the PCR product using a commercial anion-exchange PCR centrifuge column (e.g., Qiagen). 32. 32. Digest the cDNA with a suitable restriction enzyme that has a shear site on the linker (for the linkers used here, e.g. Ec0RI and EcoRV). 33. Phenol/chloroform extraction and ethanol precipitation to purify the digested cDNA. 34. 34. Ligate the DNA into a suitable vector (e.g., EcoRI or EcoRV cleaved pBluescript). 35. Transform the vector into the sensory bacteria. 36. 36. Plate culture the differential library. It is worthwhile to titrate the library first to obtain single clones. It is also important to determine the percentage of clones containing the insert and the size of the insert. The insert should be about 250 bp. If the inserted fragment is >500 bp, it can be assumed that two fragments have been inserted. 37. 37. Prepare 4 replicate blots from each starting membrane. 38. 38. Denature, neutralize and crosslink the blot according to the manufacturer's instructions. 39. 39. Hybridize these duplicate membranes with a subtractive probe. 40. 40. Pick 50-100 differentially expressed clones from the library either randomly (if the library is expected to be overwhelmingly differentially expressed) or as a result of hybridization with the probe. Prepare a small amount of plasmid DNA. 41. Sequence the insert fragments in each plasmid and group clones containing the same sequence together. 42. 42. Use RNA analysis of the starting tissue [e.g., Northern blotting, RNase protection analysis, quantitative RT-PCR, or in situ hybridization] to determine if the clones are indeed differentially expressed. Common Problems 1. Materials Double-stranded cDNA (ds cDNA) from class A and B cells. 2. Reagents ALuI and 10×ALuI buffer. RsaI 10, 15 and 75 mmol/L ATP 10 U/ul T4 polynucleotide kinase and 10×T4 polynucleotide kinase buffer Oligonucleotide Primers 3 ug/ul a1: 5'-TAG TCC GAA TTC AAG CAA GAG CAC A-3' 2.5 ug/ul a2: 5'-CTC TTG CTT GAA TTC GGA CTA-3' 3 ug/ul B1: 5'-ATG CTG GAT ATC TTG GTA CTC TTC A-3' 2.5 ug/ul B2: 5'-GAG TAC CAA GAT ATC CAG CAT-3' 10 U/ul T4 DNA ligase and 10× T4 DNA ligase buffer 40% (m/V) Polyethylene Glycol 8000 (PEG 8000) 25:24 (V/V) phenol/chloroform (balanced phenol formulation) Chloroform 5 U/ul Taq DNA polymerase and 10× Taq DNA polymerase buffer 25 mmol/L MgCl2 10 mmol/L 4dNTP Mixture Mineral oil, PCR grade, sterile 800 Ci/mmol [ α-32P ] dCTP (10 Ci/ul) Driver dNTP mixture Ethanol 1 mol/L and 5 mol/L NaCl HEPES buffer 2× subtractive hybridization buffer Streptavidin solution EcoRI and 10× EcoRI buffer or EcoRV and 10× EcoRV buffer EcoRI cut pBluescript vector EcoRV cut open pBluescript vector Transformed sensory strains 3. Consumables Radiolabeled subtraction probe 0.5 ml PCR tubes Sephacryl S-300 centrifuge column (Amersham Pharmacia Biotech) Beckman Accuspin FR centrifuge and bucket rotor or other similar centrifuge Thermal cyclers Anion exchange PCR centrifuge columns (Qiagen) 1.5 ml microcentrifuge tubes, silanized Portable Geiger counter Heating blocks For more product details, please visit Aladdin Scientific website.
ALuI RsaI ATP T4 polyribonucleotide kinase and its buffer DNA ligase and its buffer Taq DNA polymerase and its buffer Oligonucleotide primers 4dNTP mixture Drive dNTP mixture Transformation of susceptible strains HEPES buffer Streptavidin solution Differential subtractive hybridization buffer Polyethylene glycol 8000 Phenol Chloroform Chloroform Mineral oil Ethanol MgCl2 NaCl
Radioactively Labeled Differential Subtraction Probes PCR Tubes Microcentrifuge Tubes Ion-Exchange PCR Centrifuge Columns Sephacryl S-300 Centrifuge Columns Centrifuges Thermal Cyclers Portable Geiger Counters Heating Blocks