Quint Lab:arabidopsis rna isolation protocols (including siliques)
From OpenWetWareJump to navigationJump to search
Arabidopsis RNA isolation (including isolation from siliques)
(L.Oñate-Sánchez,J.Vicente-Carbajosa: DNA-free RNA isolation protocols for Arabidopsis thaliana,including seeds and siliques,BMC Research Notes 2008, 1:93)
- protocols are slightly modified and scaled up for using 80-100 mg of Arabidopsis tissues
- original protocols can be found at: http://www.biomedcentral.com/1756-0500/1/93
PROTOCOL 1 (vegetative tissues)
- Add 1200 µl of cell lysis solution (2% SDS, 68 mM sodium citrate, 132 mM citric acid, 1 mM EDTA) to ground tissue and homogenize quickly by vortexing 2 s and inverting and flicking the tube gently. Leave tubes at room temperature up to 5 min.
- Add 400 µl of protein-DNA precipitation solution (4 M NaCl , 16 mM sodium citrate, 32 mM citric acid) to the cell lysate, mix inverting the tubes gently and incubate at 4°C for at least 10 min. Spin at 4°C for 10 min.
- Transfer supernatant (s/n) to a new tube, add 1000 µl isopropanol and mix the sample by inverting gently the tube. Spin 4 min and carefully pour off the s/n. Wash pellet with 70% ethanol, air-dry RNA and resuspend in 100 µl DEPC- water.
- Add 80 µl Dnase I-Lösung (10 µl gelöste DNase + 70 µl Puffer RDD [QIAGEN RNase-Free DNase Set for on-column digestion]) Incubate 15 min at RT.
- Add 280 µl DEPC-water to the 120 µl of RNA, 100 µl of NH4Ac 7.5 M (sodium acetate can also be used) and 1600 µl 100% ethanol and mix well. Spin 20 min at 4°C, wash pellet with 70% ethanol, air-dry RNA and resuspend in 40 µl DEPC-water.
PROTOCOL 2 (seeds and siliques)
- Transfer ground tissue to a cooled 15 ml-falcon tube and quickly add 2200 µl of extraction buffer (0.4 M LiCl, 0.2 M Tris pH:8, 25 mM EDTA, 1% SDS) and 2200 µl chloroform. Vortex 10 s and keep on ice until all samples are ready. Spin for 3 min.
- Transfer supernatant (s/n) to a new tube, add 2000 µl of water-saturated acidic phenol, vortex thoroughly, add 800 µl of chloroform and spin 3 min.
- Transfer s/n to a new tube, add 1/3 volume of 8 M LiCl and mix. Precipitate at -20 °C for 1 hour (overnight at 4°C is also possible) and spin for 30 min at 4°C
- Dissolve pellet in 100 µl DEPC-water. Add 80 µl Dnase I-Lösung (10 µl DNase + 70 µl Buffer RDD [QIAGEN RNase-Free DNase Set for on-column digestion]). Incubate 15 min at RT.
- Add 1880 µl DEPC-water, 28 µl 3 M NaAc pH 5.0 and 1000 µl ethanol, mix well and spin 10 min at 4°C to precipitate carbohydrates.
- Transfer s/n to a new tube, add 172 µl 3 M NaAc pH 5.0 and 3000 µl ethanol, mix well and leave at –20°C for at least 1 hour. Spin 20 min at 4°C, wash pellet with 70% ethanol, air-dry RNA and resuspend in 40 µl DEPC- water.