Plant Molecular Biology Research Group MOLBAS, Leiden University, Leiden, The Netherlands
We have tested several methods for DNA and RNA isolation from different rice tissues (callus, cell suspensions, roots, shoots and flowers). It appears that some previously described procedures are not suitable for all tissues.
The methods of Dellaporta et al. (1983) and Rogers and Bendich (1985) gave very poor yield of DNA, which was also difficult to digest by restriction enzymes.
The methods described by Hattori et al. (1987) and by Mettler (1987) gave adequate amounts of DNA from cell suspensions but in these cases digestion of the DNA was also only partial. Using these two methods, hardly any DNA was isolated from other rice tissues.
With all four methods, RNA was isolated very poorly or was degraded.
Because we are interested to study the genomic organization of rice genes and of transferred genes in transformed rice tissues and their expression, we have developed a method for the isolation of both DNA and RNA from the same batch of tissue.
solution 1) 50 mM TRIS-HCI, 50 mM EDTA, 500 mM NaCl (pH 8.5). Sterilize and add beta-mercaptoethanol to a final concentration of 10 mM (0.698 ml to 1 liter).
solution 2) 6% (v/v) 2-butanol, 1% (w/v) tri-iso-propylnaphtalene disulfonic acid sodium salt, 2% (w/v) 4-amino-2 hydroxybenzoic acid sodium salt and 4% (w/v) sodiumdodecylsulfate (SDS). This mix is made with sterilized water.
solution 3) Sterile water.
solution 4) 1 M NaCl sterile.
solution 5) Phenol equilibrated with 1 M TRIS-HCI (pH 8.5). Normally we melt 1 kilogram phenol (at 60 deg.C) and add 1 liter TRIS buffer. After mixing, the layers are separated overnight at 4 deg.C.
solution 6) 50% phenol, 48% chloroform and 2% isoamyl-alcohol (v/v).
solution 7) 8 M LiCl sterile.
solution 8) 3M NaAcetate, sterile (made pH 5.6 with acetic acid glacial).
solution 9) 96% ethanol.
solution 10) 70% ethanol made with sterile water.
solution 11) 50 mM TRIS-HCI, 10 mM EDTA (pH 8.0), 0.1% SDS (w/v). The TRIS-EDTA solution is made and sterilized, a 20% (w/v) SDS stock solution made with sterile water is added to the desired concentration.
solution 12) 10 mM TRIS-CL, 0.2 mM EDTA (pH 8.0) sterile. It is convenient to have a sterile 50X stock (0.5 M TRIS, 10 mM EDTA pH 8.0) which can be diluted with sterile water.
solution 13) Ethidiumbromide 5 mg/ml made in sterile water.
solution 14) Isoamyl-alcohol saturated with 1OXSSC.
solution 15) CsCl/ Ethidiumbromide solution. Add to 50 ml of solution 11) 50 gram solid CsCl and 5 ml of solution 13).
Sterilize an adequate quantity of the following items: a) Mortars and pestles or warring blender of the appropiate size.
b) Centrifuge tube or bottles. We prefer to work with the Sorvall rotors SS34 or GSA. The type of tube used depends on the quantity of tissue.
c) Erlemeyer flasks of different sizes.
d) Pipets of different sizes.
The protocol is for 50 gram of wet fresh tissue. We prefer freshly harvested tissue. You can also however, harvest the tissue, freeze it in liquid nitrogen and store it at -70 deg.C some days before. In the protocol liquid nitrogen is used; if this is difficult to obtain it can be replaced by dry ice.
Step 1. Freeze the tissue in liquid nitrogen and transfer it to a warring blender (500 ml beaker), precooled with nitrogen. Add more liquid nitrogen and blend the tissue until a very fine powder is obtained.
This step can also be done in a precooled mortar with pestle, especially when small quantities of tissue (e.g. 0.5 gram) have to be processed.
Take a small sample and let it thaw, check whether the tissue is grinded very well. The extraction of the nucleic acids is optimal when the powder is very fine.
Step 2. Transfer the powder into an erlemeyer flask (1000 ml) and wait untill the material is partly thawed.
Step 3. Add 100 ml solution 1, wait until all material is thawed and solution becomes rather viscous (3-5 minutes).
Step 4. Add 100 ml solution 2, mix gently for 2 minutes, solution must become more viscous.
Step 5. Add 200 ml phenol (solution 5), mix gently for 10 minutes, take care that a homogeneous mixture is retained during this period.
Step 6. Transfer the mixture to centrifuge tubes or bottles and centrifuge 10 minutes (GSA: 5,000 rpm, SS34: 10,000 rpm).
Step 7. Transfer with a wide bore pipet the upper waterphase into a clean erlemeyer flask. Take care, not to disturb the interphase.
Step 8. Add to the waterphase of step 7 phenol (200 ml) and repeat steps 5-7.
Step 9. Add to the waterphase of step 8 phenol/chloroform (200 ml) (solution 6) and repeat steps 5-7.
Step 10. Transfer the waterphase to an erlemeyer flask (determine empty weight before) and determine the volume of the waterphase by weighing.
Step 11. Add 1/10th of the volume 3M NaAcetate (solution 8) and 2.5 volume 96% ethanol. Mix well and let stand at -20 deg.C overnight.
step 12. Transfer ethanol precipitate into centrifuge tube and centrifuge 10 minutes, 10,000 rpm (SS34) or 5,500 rpm (GSA).
Decant supernatant, taking care not to lose the nucleic acid pellet. You can centrifuge pellet on pellet to save tubes and time in later steps.
Step 13. Wash pellets with 70% ethanol: GSA bottle with 100 ml, SS34 tube with 30 ml. Mix, let stand for 5 minutes, centrifuge 5 minutes, 10,000 rpm (SS34) or 5,500 rpm (GSA). Decant supernatant, repeat this washing step at least two more times.
Step 14. Dry pellets as briefly as possible in vacuum desiccator.
Step 15. Dissolve pellet in 6 ml TES (solution 11). This takes at least 5 hours. If high molecular weight DNA is required do not vortex and avoid shearing of DNA by small bore pipets. It is important that all nucleic acids are well dissolved otherwise much DNA will coprecipitate with RNA in the next step.
Step 16. Add 2 ml 8M LiCl (solution 7), so that final concentration is 2M. Mix well and let stand for at least 8 hours at 4 deg.C.
Step 17. Centrifuge 15 minutes, 10,000 rpm (SS34) at 4 deg.C.
Step 18. Decant the DNA containing supernatant in a preweighed tube.
RNA preparation: Step 19. Wash the RNA pellet 3 times with 70% ethanol (see step 13).
Step 20. Dry the pellet briefly in vacuum desiccator.
Step 21. Dissolve the RNA pellet in 1 ml TES (solution 11) and measure the RNA concentration, we usually dilute 25 ul of the RNA solution into 1 ml. If RNA has to be completely DNA free, repeat LiCl precipitation (step 16- 21) once or twice or even better: Treat with proteinase K or pronase which have been predigested and treat with RNase free DNase.
DNA preparation: Step 22. Determine the volume of the DNA containing supernatant of step 18 by weighing. Add exactly 1 gram of solid CsCl per ml DNA solution. Mix gently until the salt is dissolved.
Step 23. Add 0.1 ml of the ethidiumbromide solution (solution 13) for 1 ml of original DNA solution. Mix well and transfer to an ultracentrifuge tube suitable for a Beckmann Type-65 rotor. If you use sealtubes, you can fill the remainder with solution 15. Otherwise use oil to fill tubes completely. Centrifuge at 45,000 rpm for 42-72 hours at 20 deg.C. Often the DNA is already visible in ordinary light. Use long wave ultraviolet illumination to collect the DNA. This is done by first making a hole in the upper part of the tube or by removing the cap of the tube, and by inserting a 18 Gauge (inner phi 1.5 mm) needle attached to a syringe into the side of the tube just below the DNA band.
Step 24. If the gradient is very dirty and a lot of other material is present at the same level as the DNA band, a second CsCl gradient has to be run. The DNA of step 23 is transferred to a new tube, tube is filled with solution 15, and centrifuged again for 42-72 hours.
Step 25. The ethidiumbromide is removed from the DNA solution as follows: a. add an equal volume isoamyl-alcohol (solution 14)
b. mix the two phases (not too vigorous, to prevent shearing of DNA)
c. let stand to separate the two phases, or centrifuge for 2 minutes at 2000 g
d. remove the upper isoamyl-alcohol phase with a pipet
e. repeat steps a-d until all pink colour disappears from the DNA lower phase
f. if a cloudy interphase arises, add one or two drops of water, mix again, and go on. Prevent to pipet off this cloudy interphase, since your will lose DNA.
Step 26. Dialyze the colourless DNA phase against solution 12. Change several times the buffer until no isoamyl-alcohol smell can be detected. Refresh once more the buffer and dialyze for 2 more hours. Collect DNA from dialysis bags and measure OD at 260 nm.
Fig. 1. 5 pg DNA from different rice tissues was cut with BamHI, and separated on a 0.7% agarose gel, stained with ethidiumbromide (left panel) and transferred to Gene- ScreenPlus according to Southern (1975). After UV crosslinking the filter was hybridized with a rice cDNA probe specific for green tissues (gene 5; B.S. de Pater et al. submitted). Lane 1 contains marker DNA mixed with DNA isolated from leaves of two weeks old T309 plantlets (lane 8). From 10 days old T309 seedlings DNA was isolated from leaves (lane 2-3), nodes (lane 4) and roots (lane 5). DNA was also isolated from two different T309 cell suspensions (lane 6 and 7).
The yield of DNA /gram wet weight is about 70 ug for cell suspensions, 20- 50 ug for 10 day old seedlings and 10 pg for old leaves. Roots generally yield half the quantity of the green parts. DNA isolated using this method, was used to construct several rice genomic banks (Hensgens et al., in preperation) and has been used to study the organization of several rice genes (see figure 1) and to analyse transformed rice tissues.
RNA yields are in general 10 times higher than the DNA yield. When very small quantities of tissue are available, yield can be increased 2-5 times by reextracting all phenolphases subsequently with 0.5 volume 1M NaCl (solution 4) and 0.5 volume water. A better yield is also obtained by increasing the ratio between the volumes of the buffers and weight of the tissue. This will however double the labour.
The RNA, isolated in this way, gives very good results with Northern blot analysis and isolation of poly A' RNA. For the synthesis of intact cDNA it is however recommended to treat the RNA fraction with DNase as described in step 21.
Dellaporta, S. L., J. Wood and J. B. Hicks, 1983. A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter 1(4): 19-21.
Hattori, J., S. G. Cottlob-Mc Hugh and D. A. Johnson, 1987. The isolation of high-molecular- weight-DNA from plants. Analytical Biochemistry 165: 70-74.
Mettler, I. J., 1987. A simple and rapid method for minipreparation of DNA from tissue-cultured plant cells. Plant Molecular Biology Reporter 5(3): 346-349.
Rogers, S. O. and A. J. Bendich, 1985. Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tisse. Plant Molecular Biology 5: 69-76.
Southern, E., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.