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Protocol

Isolation of total RNA from mammalian cells

PK Buffer - see below Lysis Buffer - see below 7.5 M NH4OAc (sterile) DEPC treated H20 DEPC treated 3 M NaOAc Phenol:CHC13:isoamyl alcohol (24:24:1) TE (pH 7.5) sterile Cold PBS Needles Syringes DNAse I (Worthington DPRF) Lysis Buffer: 140 mM NaCl 1.5 mM MgCl2 10 mM Tris-HCl pH 8.6 0.5% Nonidet P-40 (BDH) 2x PK Buffer: 200 mM Tris-HCl pH 7.5 25 mM EDTA 300 mM NaCl 2 % SDS 400 ug/ml proteinase K - Add just before using To Isolate Cytoplasmic RNA Only: Wash cells with ice cold PBS Scrape cells into tube. Falcon 1059 Spin down. 5K- 5 mins. in SS34 Resuspend in 1 ml lysis buffer Let sit 5 mins. on ice Underlay with 3 mls of lysis buffer containing 24% (W/V) sucrose 1% NP40 Centrifuge 8000 rpm at 40C for 20 mins in Sorvall HB4 rotor. (can use Falcon 2059 tubes) Remove top layer. This contains cytoplasmic RNA. Mix with equal volume of 2x PK buffer and continue RNA prep. DNAse step is now optional for "crude" Northerns The pellet from centrifugation is the nuclei and can be used to isolate nuclear RNA or DNA
1. Aspirate off media, wash calls with 3 mls. cold PBS 2. Add 1 ml. of 50:50 mixture of Lysis Buffer: PK Buffer (200 ug/ml). 3. Scrape lysate into sterile plastic tubes using rubber policeman. Use Falcon 1059 tubes. 4. Start incubating at 370C and shear lysate 5 times with a 21 gauge needle. 5. Total incubation >30 minutes. 6. Extract once with r)henol:CHC13 (1:1), spin. Place aqueous phase in new tube (Falcon 1059). 7. Add 1/15 volume 7.5 M NH4OAc, two volumes ethanol. Mix. 8. Spin, 9K -in SS34 rotor 10 mins. Wash pellets with 70% ethanol. 9. Resuspend in 375 ul TE (pH 7.5). Transfer to Eppendorf tube. Add 2 ul 1 M MgCl2 20 ul vanadyl ribonucleotide complex (optional) 10. Add 4 units DNAse I (DPRF grade - Worthington) or Promega 11. Incubate 370C, 301 12. Phenol:CHC13 extract. Add .35 ul 7.5 M NH4OAc, 2 volumes ETOH 13. Mix, spin 3 mins in Eppendorf. Remove supernatant. 14. Resuspend in 3 M NaOAc. Vortex vigorously to redisperse pellet. 15. Repellet by spinning 3 mins. Wash with 70% ethanol. ifusing vanadyl ribonocleotides pellet must be white. If still gray repeat step 14. 16. Resuspend in 460 ul DEPC treated H20. Reserve 10 ul for UV absorption. A260 @ 1 = 40 ug/ml RNA. 17. Add 35 ul 7.5 M NH4OAc, two volumes ETOH to remaining. 18. Store as ethanol mixture. To use RNA, vortex tube very hard to disperse precipitate and remove required amount to pellet separately.
Favaloro et al. Methods in Enzymology 65:718-749,1985 ISOLATION OF TOTAL RNA FROM MAMMALLIAN CELLS

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