NO Beads Revised version 8-27-99 PROTOCOL FOR SINGLE CELL mRNA AMPLIFICATION/ REVERSE NORTHERN ANALYSIS I. First round amplification A.First strand cDNA synthesis 1.Add to single cell: Cell contents plus recording solution 1x RT buffer dGTP (10mM) dATP (10mM) dTTP (10mM) dCTP (10mM) Oligo-dT-T7(100ng/ul) DTT(100mM) RNasin AMV-RT(25U/ul) total volume 2.Mix gently; incubate at 42C for 90min. 3.Phenol/chloroform extraction cDNA: DD-H2O 3M Na acetate chloroform buffer-saturated phenol final volume 105ul 15ul 75ul 75ul 300ul 3ul 17.5ul 1ul 1ul 1ul 1ul 1ul 3ul 0.5ul 1ul 30ul 4.Vortex for 10 sec. Centrifuge for 3 min. Carefully remove~145ul aqueous (top) phase to a new tube. 5.Precipitate with ethanol. Add 300ul 100% ice-cold ethanol,1ul tRNA(5ug) 6.Leave on dry ice for least 20-30 mins. . Centrifuge at 18,000rpm at 4C for 30 min. B.Second strand cDNA synthesis 7. Dry & resuspend pellet in 20ul DDH2O. 8.Heat at 90-95C for 3 min to denature RNA:DNA hybrid. 9. Cool quickly on ice; centrifuge briefly to bring down condensation. 10.Add to sample: (20ul) 10x 2nd strand buffer 100mMDTT 4 dNTPs(2.5mM each) random hexamers (100ng/ul) T4 DNA polymerase (5U/ul) Klenow (5U/ul) DDH2O 5ul 2ul 5ul 1ul 0.2ul(1U final) 0.5ul(2U final) 16.3ul final volume 50ul 11.Mix gently; incubate at 14C for a minimum of 5 hrs, or overnight. C.Blunt-end treatment 12. S1 nuclease cut or blunt –end repair (if go into reverse northern): dilute 1ul of S1 nuclease in 400ul of 10x S1 buffer. 13.Add to 2nd strand: DDH2O 400ul 10x S1 buffer 50ul diluted S1 nuclease (1U) 1ul tRNA 1ul 14. Incubate at 37C for 5 min. 15. Extract with phenol/chloroform(0.5x): add to blunt-end treated 2nd strand sample 452ul chloroform 226ul buffer-saturated phenol 226ul 16. Vortex 10 sec. centrifuge for 3min. extract top aqueous layer into a new tube. 17.Precipitate with 1ml 100% ETOH on dry ice for 20-30 mins. 18.Centrifuge 18,000rpm @4C for 30 min., dry pellet. 19.Resuspend pellet in 20ul of DDH2O, add DD-H2O 10x KFI buffer 100mM DTT* 4 dNTPs T4 DNA polymerase(5U/ul) 21ul 3ul 3ul 3ul 0.2ul final volume 50ul *replace with DD-H2O if already included in the 10xKFI buffer 20.Incubate at 37C for 15-30 min. 21. Phenol-chloroform extract ds-cDNA. Add: DD-H2O 3M sodium acetate chloroform buffer-saturated phenol 85ul 15ul 75ul 75ul final volume 300ul 22.vortex for 3 sec. Centrifuge for 3 mins. Carefully transfer ~145ul top aqueous layer to a new tube. 23.Add 300ul 100% ice-cold ETOH on dry ice for 20-30mins.Centrifuge at 18,000rpm at 4C for 30 mins. 24.Resuspend pellet in 20ul of DD-H2O. Drop dialyze 10-20ul sample against 50 ml DD-H2O for at least 4 hrs. D.First round aRNA amplification (cold reaction) 25. use 1/10 of total sample & add: Dialyzed sample 10x TSC buffer(RNA amplification buffer) 20mM spermidine* 4rNTPs(with UTP)(2.5mM each) 100mM DTT RNasin 0.5ul T7 RNA polymerase (1000U/ul) DD-H2O 2.0ul 2.5ul 2.0ul 2.5ul 1ul 1ul (1000U final) 8.5ul final volume 20ul *replace with DD-H2O if already included in the 10x RNA amplification buffer. 26. Mix gently; incubate @ 37C for 4 hrs. 27.Phenol/chloroform extract aRNA. Add to tube : DD-H2O 3M ammonium acetate* chloroform buffer-saturated phenol 95ul 15ul 75ul 75ul final volume 300ul *ammonium acetate if more efficient than sodium acetate at removing free NTPs. Adding salt at this step(instead of subsequent ethanol step) improves separation of the aqueous and organic phases. 28. Vortex for 10 sec. Centrifuge for 3 min. Carefully remove 145ul aqueous (top) layer to a new tube. 29. Add tRNA* (1ug/ul) 1ul 100% ice-cold ethanol 450ul *use of a carrier is highly recommended to aid precipitation of miniscule amounts of RNA (especially from single cells.) However, tRNA can sometimes cause artefacts in PCR reactions. While this is usually not a problem wtih routine PCR analysis, it may be critical when doing differential display-PCR, for example. Glycogen is also thought to interfere with many manipulaitons. Other carriers have not been tested, but may be appropriate in these cases. 30.Precipitate on dry ice for 20-30mins. Centrifuge 18,000 @ 4C for 30 mins. Resuspend the pellet in 19.5ul of DD-H2O. II. Conversion of aRNA to double-stranded cDNA A. First strand cDNA synthesis 31. Denature 19.5ul aRNA sample at 90-95 C for 3 min. 32.Quick cool, quick spin to bring down condensation. 33.Add to denatured aRNA sample: 5x first strand buffer 4 dNTPs (2.5mM each) random hexamer (100ng/ul) 100mM DTT RNasin Superscript RT final volume 34.Mix gently; incubate @ 42C for 90 min. 35.Phenol-chloroform extract ss-cDNA. add: DD-H2O 3M sodium acetate chloroform buffer-saturated phenol final volume: 105ul 15ul 75ul 75ul 300ul (19.5ul) 3.0ul 3.0ul 1.0ul 2.0ul 0.5ul 1.0ul 30ul 36.Vortex for 10 sec. Centrifuge for 3 min. Carefully transfer 145ul aqueous (top) layer to a new tube. 37.Add 300 ul 100% ice-cold ethanol to precipitate on dry ice for 20-30 mins. 38. Centrifuge at 18,000rpm at 4C for 30 min. Resuspend the pellet in 12.3ul of DDH2O. B. Second strand cDNA synthesis 39.Heat at 90-95 C for 3 min to denature aRNA:DNA hybrid. 41. Quick cool, quick spin to bring down condensation. 42.Add to sample: 10x KFI buffer 100mM DTT* 4 dNTPs(2.5mM each) T7-oligo(dT)24 primer(100ng/ul) T4 DNA polymerase (5U/ul) Klenow (5U/ul) 2ul 2ul 2ul 1ul 0.2ul (1U final) 0.5ul (2U final) final volume: 20ul *replace with DD-H2O if already included in the 10x KFI buffer 43. Mix gently; incubate at 14C overnight. 44.Centrifuge briefly to bring down condensation. C. Blunt-end reaction 45. S1 nuclease cut or blunt –end repair (if go into reverse northern): dilute 1ul of S1 nuclease in 400ul of 10x S1 buffer. 46.Add to 2nd strand: DDH2O 400ul 10x S1 buffer 50ul diluted S1 nuclease (1U) 1ul tRNA 1ul 47. Incubate at 37C for 5 min. 48. Extract with phenol/chloroform(0.5x): add to blunt-end treated 2nd strand sample 452ul chloroform 226ul buffer-saturated phenol 226ul 49. Vortex 10 sec. centrifuge for 3min. extract top aqueous layer into a new tube. 50.Precipitate with 1ml 100% ETOH on dry ice for 20-30 mins. 51.Centrifuge 18,000rpm @4C for 30 min., dry pellet. 52.Resuspend pellet in 20ul of DDH2O, add If planning to do reverse Northerns, you may wish to blunt-end to eliminate possible”wrap-around” RNA synthesis, which could reduce (via RNA-RNA selfhybridization)the amount of aRNA probe available for hybridization to cDNA on the blot. 53.Add to 2nd strand reaction: DD-H2O 10x KFI buffer 100mM DTT* 4 dNTPs(2.5mM each) T4 DNA polymerase (5u/ul) (20ul) 21ul 3ul 3ul 3ul 0.2ul final volume 50ul *replace with DDH2O if already included in the 10x KFI buffer 54. Incubate at 37C for 15-30min. 55. Phenol-chloroform extract ds-cDNA. add DD-H2O 3M sodium acetate chloroform buffer-saturated phenol final volume: 85ul 15ul 75ul 75ul 300ul 56. Vortex for 10 sec.Centrifuge for 3min. Carefully transfer 145ul (top) aqueous phase to a new tube. 57.Add 300ul 100% ice-cold ethanol to precipitate on dry ice for 20-30mins. 58. Centrifuge @18,000rpm at 4C for 30 min. Resuspend the pellet in 20ul of DD-H2O. At this point the sample may be used for PCR analysis in a 1:100 final dilution. III. Second round aRNA amplification (hot reaction) 51.Drop-dialyze 10-20ul sample against 50ml DD-H2O for at least 4 hrs. 52. Combine: 1/10 dialyzed sample plus DD-H2O 7.7 ul 10x RNA amplification buffer 20mM spermidine* 100mM DTT 3 rNTPs(ATP,GTP,UTP)(2.5mM each) 100uM CTP 2.0ul 2.0ul 1.0ul 2.0ul 0.8ul RNasin 0.5ul alpha-[32P}-CTP(3000Ci/mmol;1mci/100ul 3.0ul(4mM final=80pmol) -remove 0.5ul and spot onto 1MM whatman paper(=TCA before) add T7 RNA polymerase (1000U/ul) final volume 1.0ul 20ul *replace with DD-H2O if already included in the 10x RNA amplification buffer 53.Mix gently; incubate at 37C for 4-8 hrs. (6 hrs. is recommended) Prehybridize blots (go to step 57 ). 54. Centrifuge briefly to bring down condensation. 55.Remove 0.5ul & spot onto 1MM Whatman paper (=TCA after) 56.TCA precipitation a.Wash “TCA-before” & “TCA-after” samples in 10% TCA for 5 min on a rotating plateform. Allow a sufficient volume of TCA for the samples to flow freely. b.Replace with fresh 10% TCA and wash for 5 min. Wash once more for 20 min. c.Air dry before measuring radioactivity levels, either by counting in a scintillation counter or by listening with a Geiger counter. There should be a significant increase in the amount of radioactivity measured in the “TCA after” samples relative to the “TCA before” samples, signifying successful a RNA synthesis as measured by incorporation of 32P-CTP. You should be able to detect an audible difference using a Geiger counter. IV. Reverse Northern Blotting A. Prehybridization 57.Prehybridization solution: ultrapure formamide 20x SSC 50% dextran sulfate 50x Denhardt’s salmon sperm DNA Total 50ml 20ml 20ml 10ml 1ml 101ml final concentration 50% 4x 10% 5x 100ug/ml 58.Place blot(s) into a 15ml or 50ml conical tube(cDNA side toward the lumen of the tube).Allow sufficient room to avoid overlapping membrane (unless using nylon mesh), while minimizing the volume of prehyb solution needed. Using a minimal volume will result in a higher concentration of probe and better hybridization. 59.Add prehyb solution to blots. 4ml per 15ml tube or 8 ml per 50ml tube is sufficient. Thoroughly wet the membranes and remove large bubbles between the membrane(s) and the side of the tube. 60. Prehybridize for at least 3 hrs at 42C in the hybridization oven. When using 50ml conicals, it is recommended that the tubes be sealed with parafilm to avoid leakage. B.Hybridization 61.Heat aRNA probe at 90-95C for 5 min. 62.Cool quickly on ice;centrifuge briefly to bring down condensation. 63.Keep all samples on ice to minimize renaturation. 64.Add the probe to the prehyb solution in the tube containing the blot(s).Do NOT Let the probe come in direct contact with the blot. 65.Recap the tube(and re-parafilm); mix well before returning to 42C oven. 66.Hybridize for at least 16 hrs. when using dextran sulfate (otherwise 2 days) C.Washing 67.After hybridization, remove blots directly into a large Tupperware container with 500-800ml of 2x SSC, 0.1% SDS. Place on a rotation platform to wash for 30 mins. at room temp. 68. Remove wash. Do a second wash in 2x SSC, 0.1% SDS for 30 min. 69.Remove second wash. Add 0.2x SSC, 0.1%SDS & wash for at least one hour. D.Autoradiography 70. Remove blots from third wash solution and place directly into plastic sealable bags. Keep the blots moist in case more washing is required, or if you desire to strip the blots & reuse them. 71. Seal the plastic bag. Expose the blots to X-ray film or phosphorimaging screen. APPENDIX: A. Amplification Buffers (for 50 ml in DEPC-H2O): 10x RT 500mM Tris-base, pH 8.3 1.2M KCl 100mM MgCl2 filter. Store in 1ml aliquots at -20 °C. 10x 2nd Strand 1M Tris-base, pH 7.4 200mM KCl 100mM MgCl2 400mM (NH4)2SO4 filter. Store in 1ml aliquots at -20 °C. 50mM DTT 10x RNA Amplification 400mM Tris-base, pH 7.5 70mM MgCl2 100mM NaCl filter. Store in 1ml aliquots at -20 °C. 20mM spermidine 10xKFI 200mM Tris-base, pH 7.5 100mM MgCl2 50mM NaCl filter. Store in 1ml aliquots at -20 °C. 50mM DTT 3.03g (pH with HCl) 4.47g 1.02g Mix. Filter through 0.2µm 6.06g (pH with HCl) 0.746g 1.02g 2.64g Mix. Filter through 0.2µm add later* 2.42g (pH with HCl) 0.712g 0.292g Mix. Filter through 0.2µm add later* 1.21g (pH with HCl) 1.02g 0.146g Mix. Filter through 0.2µm add later* *The buffer can be stored longer (for years) without this component. C. Preparation of cDNA blot 1. Linearize cDNAs of interest using an appropriate restriction enzyme. Check for complete digestion before proceeding. There is no need to ethanol-precipitate the cDNAs. The integrity of linearized plasmid that has been stored for more than a couple weeks .should be rechecked on a gel before proceeding. Remember to include linearized plasmid vector as a background control. 2. DEPC- treat the top slotted portion of the blotting apparatus (Milliblot-S System). 3. Cut 3 pieces of 3MM Whatman paper and one piece of nitrocellulose or nylon membrane to fit within the rubber gaskets of the apparatus. 4. Using clean gloves or DEPC-treated forceps, wet the Whatman papers and membrane in 10x SSC. 5. Assemble the apparatus from the bottom. Place the three sheets of Whatman paper on top of the middle slotted piece, then the membrane. Make sure that the paper and membrane stay within the gaskets to ensure a tight seal. Roll out any air bubbles that may have formed between the layers before placing the top slotted piece onto the apparatus and completing the assembly. Add 10x SSC to the wells to moisten and set aside. If using vacuum application, check at this time to ensure that suction is gentle enough (ie. at least 5 min for 100µl volume) before loading samples. 6. Prepare 0.5 µg of linearized cDNA in 100µl 10x SSC per well. 7. a. b. c. d. Heat denature samples at 85-90°C for 5 minutes. Cool quickly on ice. Centrifuge briefly to bring down condensation. Keep the samples on ice while loading. 8. Remove excess 10x SSC from the wells of the blotting apparatus (Shake vigorously into the sink). Add 100 µl sample to each well. It is a good idea to carefully write out the layout of cDNAs before loading and to note any deviations immediately after loading. 9.Apply a gentle vacuum to draw samples through the slots. This should take at least 5 minutes or you risk pulling the samples through the membrane. Alternatively, one can use gravity to draw the samples through (although this will take several hours). 10. Once samples have been drawn through and the wells are completely dry, disassemble the apparatus. Take note of the orientation of the blot (perhaps cut a corner) before removing the membrane and placing it on Whatman paper to dry. 11. UV-crosslink the cDNA to the membrane using the Stratalinker. D. Genome Systems protocol for stripping blots 1. 2. 3. 4. Place filters in hyb bottle with DNA side to the lumen. Add 50 ml of 0.4M NaOH for 45 min at 45 °C and 20 rpm. Rinse once with 50 ml of 0.2M Tris-HCl, pH 7.2, 0.1% SDS, 0.1x SSC. Wash two times in 100 ml of 0.2M Tris-HCl, pH 7.2, 0.1% SDS, 0.1x SSC for 10 min at room temp. and 20 rpm 5. Reimage moist filters to ensure stripping was complete. 6. If stripping was not complete, repeat process with 100 ml of 0.4M NaOH prewarmed to 50 °C for 1 hour at 50 °C and 20 rpm. 7. Repeat washes as above.