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m6f from proteinase-K-SDS-digested cell lines and tissues
This method can be used to isolate mRNA directly from cultured cells by adding the oligo (dT) cellulose to proteinase K-digested crude cell lysate. Some whole tissues can be lysed directly in the proteinase K solution, whilst others, notably pancreas, need prior extraction of total RNA using guanidinium or lithium-urea. The total RNA can then be PolyA+ selected using oligo(dT) cellulose
Day 1 Homogenise tissues and isolate mRNA
Day 2 Recover mRNA, quantitate and formaldehyde-agarose gel check
Reagents All reagents must be made with sterile MilliQ water. Use
DEPC with care - it may inhibit subsequent enzyme reaction Sterile MilliQ water 10 M NaOH Proteinase K at 10 mg / ml in sterile water Oligo(dT) cellulose (Boehringer or equivalent) STE solution 100 mM NaCl, 10 mM TrisCl pH 7.4 and 1 mM EDTA pH 8.0 20% SDS in sterile water 5 M NaCl 1 M TrisCl. pH 7.4 (DEPC is unstable in Tris buffers) 500 mM EDTA pH 8.0 Binding (high salt) buffer make up just before use : the SDS will gradually precipitate out at room temperature : 500 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA pH 8.0, 0.5% SDS Wash buffer 100 mM NaCl, 10 mM TrisCl pH 7.4, 1 mM EDTA, 0.5% SDS. Elution buffer 10 mM TrisCl pH 7.4, 0.1 mM EDTA pH 8.0, 0.5% SDS Acid phenol-chloroform. 3M sodium acetate pH 6.0
In advance 1 Grow cell cultures
2 Prepare total RNA from adult organs if desired 3 Clean Polytron probe with 3 changes of distilled water.
If the RNA is to be used for PCR, rinse in 0.25M HCl for 15 minutes at room temperature to depurinate all contaminating DNA
4 Prepare the oligo(dT)cellulose
Wash 1 g of oligo(dT)cellulose in 50 ml of 400 mM sodium hydroxide in sterile water for 30 minutes at room temperature on a rotating wheel. Neutralise by washing twice in 50 ml of 500 mM TrisCl pH 7.2, followed by 6 changes of sterile water. Wash in 50 ml of binding buffer to equilibrate the salt concentration, recover by centrifugation and then resuspend in 50 ml of binding buffer
Day 1 5 Resuspend total RNA in up to 40 ml of 100 M NaCl, 10 mM TrisCl pH 7.4, 1 mM EDTA pH 8.0 and 0.5% SDS with 300 mg / ml proteinase K in a sterile 50 ml tube. Incubate for 30 - 60 minutes at 65oC 6 Homogenise cultured cells or harvested organs in up to 40 ml of 100 M NaCl, 10 mM TrisCl pH 7.4, 1 mM EDTA pH 8.0 and 0.5% SDS with 300 mg / ml proteinase K in a sterile 50 ml tube. Incubate for 30 - 60 minutes at 65oC 7 Adjust the NaCl concentration to 500 mM and add an appropriate amount of oligo(dT) cellulose in binding buffer 1 and the RNA sample allowed to bind for 2 - 4 hours at room temperature in a total volume of 50 ml on a rotating wheel 8 To remove unbound (mainly ribosomal) RNA, spin the RNA - oligo(dT)cellulose mix at 5 000 rpm for 5 minutes, decant the supernatant and resuspend in 25 ml of washing buffer warmed to 37oC. Mix on the rotating wheel for 15 minutes 9 Recover the RNA - oligo(dT)cellulose mix and wash again 25 ml of washing buffer warmed to 37oC. Mix on the rotating wheel for 15 minutes 10 Spin the RNA - oligo(dT)cellulose mix at 5 000 rpm for 5 minutes, decant the supernatant and recover bound poly(A+)RNA by eluting twice with 5 ml of 0.5% SDS in sterile water heated to 65oC. 11 Extract the eluate once with acid-phenol-chloroform. This removes any enzymatically active proteinase K and also any oligo(dT) cellulose that has been carried into the supernatant 12 Precipitate the RNA with one volume of isopropanol and 0.1 volumes of lithium chloride 2 and recover by centrifugation at 1200g for 10 minutes 13 Wash the pellet in 70% ethanol and then resuspend in 50 - 500 ml of 2 mM DTT, 1 u / ml RNasin in sterile Milli Q water 14 Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm
Day 2 15 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis 16 Store the RNA at -70oC . 17 Regenerate the oligo(dT) cellulose. Wash it well in several volumes of elution buffer, re-treat with 400 M NaOH as in 4, spin down and resuspend in 500 mM TrisCl pH 7.4. Wash for 15 minutes, spin down, check the pH of the supernatant and resuspend again in 500 mM pH 7.4 if it is still alkaline. Resuspend the neutralised oligo(dT)cellulose in sterile water, wash to remove the salt, spin down and resuspend in absolute ethanol. Store at -20oC protected from light
Notes 1 I have successfully isolated spleen, liver, macrophage and eye RNA using direct lysis in proteinase-K solution. Use large volumes (50 ml) and only a few organs 2 Brain, liver and spleen have a tendency to degrade and it is best to isolate total RNA first. Pancreas does not work at all with this method : it must first have the RNase denatured and then removed before poly(A+ ) selection. 3 Pancreas total RNA is best proteinase-K digested before oligo(dT) selection to remove any residual undenatured RNaseA. Other total RNAs can be safely resuspended directly in binding buffer without proteinase digestion. 4 The whole procedure can be performed in Econo columns. However, it is slower and the resulting mRNA has more rRNA contamination in my hands than using the 50 ml tubes 5 Recover very small amounts of PolyA+ with 20 mg of tRNA and collect in the ultracentrifuge 6 To remove any DNA, resuspend the pellet at 13 in 10 mM TrisCl pH 7.4, 1 mM EDTA, 10 mM MgCl2, and add 1 unit of RQ1 RNase-free DNaseI (Promega) per 10 ml volume. Incubate at 37oC for 30 minutes. Stop the reaction with 10 mM EDTA and 0.2% SDS. Extract once against acid-phenol, re precipitate and recover by centrifugation.
Reference #2 T. J. Gonda, D. K. Sheiness and J. M. Bishop (1982) Transcripts from the cellular homologs of retroviral oncogenes : distribution among chicken tissues Mol Cell Biol 2:617 - 624
Reference #462 Aviv and P. Leder (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose Proc Natl Acad Sci U S A 69:1408-12
Reference #468 M. J. Elliott, B. E. Faulkner-Jones, H. Stanton, J. A. Hamilton and D. Metcalf (1992) Plasminogen activator in granulocyte-macrophage CSF transgenic mice J. Immunol. 149:3678 - 3681
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