Pikaard lab plant RNA/DNA isolation protocol
this protocol allows you to isolate both genomic DNA and total RNA from the same tissue sample
1. Harvest plant material and freeze using liquid nitrogen. Store at -80°C. until ready to isolate RNA and DNA.
2. Place Mortar wrapped in foil on ice. Pre-chill all tools in liquid nitrogen including mortar, pestle and spatula. Add ~2 g plant material to grind (as little as 0.25g works fine also without modifying the protocol). Grind plant material to a fine powder adding liquid nitrogen as necessary to avoid thaw. Transfer plant powder to a 15 ml snap cap Falcon tube containing 4 ml of RNA/DNA extraction buffer and vortex immediately.
3. Add 3 ml phenol and vortex 15 sec. Add 3 ml chloroform/isoamyl alcohol (24:1 v/v) and vortex 15 sec. Centrifuge 10 min at 5000 rpm (~3000 x g) in Beckman JA-20 rotor using rubber adaptors for 15 ml Corex tubes. Transfer aqueous phase (~6ml) to a new 15 ml snap cap tube. Repeat the phenol/chloroform (1:1) extraction and centrifugation. Transfer aqueous phase (~6ml) to a new 15 ml snap cap tube containing 8 ml isopropanol to precipitate total nucleic acids. Mix well and store in -20°C freezer at least 15-30 minutes (overnight is often convenient).
4. Centrifuge at 7000 rpm (~6000 x g), ~30 minutes, 4°C in Beckman centrifuge JA-20 rotor, using rubber adaptors, to pellet the nucleic acids (RNA and DNA). Pour off supernatant and drain tubes by inverting on paper towel. Resuspend pellets in 500 ul DEPC-treated ddH2O by pipetting up and down repeatedly. Centrifuge at 3000 rpm 5 min. to pellet insoluble material. Transfer supernatant to a 1.7 ml eppendorf tube containing 500 ul 4M LiCl, mix well and incubate 3 hrs. to overnight on ice or in cold room.
5. Spin at top speed in microfuge, 15 minutes (room temperature is fine) to pellet the large RNAs that precipitate in LiCl. Small RNA, e.g. tRNAs, and DNA remain in the supernatant and can be recovered by ethanol precipitation).
6. To isolate the DNA, transfer 500 ul of the supernatant from step 5 to each of two 1.5 ml microfuge tubes. Add 1 ml EtOH to precipitate DNA (no additional sodium or ammonium actetate needs to be added due to the high salt concentration). Incubate on ice 5-15 minutes, centrifuge at top speed to pellet the DNA and wash the pellet with 70% EtOH. Dry pellet and resuspend each in 150ul H2O. Combine resuspended DNAs into one tube and spin 2 minutes to remove insoluble material. Transfer supernatant to a fresh tube. Add 150 ul of 7.5 M ammonium acetate, mix, and add 900 ul absolute ethanol to precipitate DNA a second time. Incubate on ice 5-15 minutes, spin to pellet DNA, wash with 70% ethanol and dry pellet. Resuspend in 100 ul water. Because there is a substantial amount of small RNA in these samples, DNA concentration is best estimated by running an agarose minigel and comparing the ethidium-bromide signal versus a known standard, such as lambda DNA. Typical concentrations are 200-400 ng of high molecular weight DNA per microliter. You should see a tight band, not a smear.
7. RNA isolation - Resuspend pellet from step 5 in 200 ul DEPC ddH2O. If necessary, spin to remove insoluble material. Take A260 and A280 readings using 5 ul RNA diluted into 1 ml H2O. Calculate RNA yield based on the estimate that 1 A260 unit=40 ug/ml. Don't forget your dilution factor (200). Store RNA in aliquots at -20°C or -80°C.
8. The RNA is ready to use for primer extension analysis, S1 nuclease protection or running on agarose formaldeyde gels (which we always do to assess the quality of the RNA-sharp ribosomal RNA bands on an ethidium-stained gel are a good sign of quality; a smear is a bad sign).
Before using RNA for RT-PCR treat RNA with DNase RQ1: Mix 25 ug RNA with 5 units DNase RQ1 and 20 units RNasin in 90 ul 1x NEB (New England Biolabs) buffer 2. Incubate 1 hr. at 37°C. Phenol/chloroform extract, EtOH precipitate, 70% EtOH wash, dry pellet and resuspend in 25 ul DEPC-treated ddH2O. Store at -80°C until ready to use.
RNA/DNA extraction buffer (50 ml)
250 mM Tris-Cl pH 8.5 12.5 ml 1 M
375 mM NaCl 3.75 ml 5 M
25 mM EDTA 2.5 ml 0.5 M
1% SDS 5.0 ml 10%
1% b-mercaptoethanol 0.5 ml 100%
25.75 ml H2O (to 50 ml)