DNAse-free RNA isolation (for qRT), the Citrate-Citric Acid method
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plant RNA isolations often requires the of use unhealthy organic solutions like phenol or beta-mercaptoethanol, and although I still prefer the TriZOL method or the SDS-TriZOL combo for when I need large quantities of great RNA, you have to wonder if it will ever be needed for me to do a northern blot. This method described here is for when you dont need that much RNA, for example because you’re going to do qRT-PCR. Here is a method to quickly and more importantly cheaply isolate good RNA from relatively small amounts of tissue (30-50 mg/a few 0.5 cm leaf discs) using only a low pH from citric acid to inactivate the RNAses. The method is described by Oñate-Sánchez and Vicente-Carbajosa (2008).
I like it much better than the plant RNA kit from QIAgen, with those little columns and wash buffers that completely make u lose the appreciation for what ur doing. It takes about the same time, but with less steps, and at least in my hands the yield is much better. It costs about as much as some citric acid, sodium citrate and NaCl, if u dont already have a couple big containers with that stuff gathering dust on the shelves, so u can use your money for the RNAse free DNAse and qRT-enzymes. So far, I have used it for Arabidopsis and Alfalfa
I think DEPC treating of water is not really needed anymore. If you have a Milli-Q machine, the water comes out RNAse free, seriously. So dont buy RNAse free water from Ambion, thats a waste of money. Also, autoclaving or baking your mortar and pestles is a complete waste of time they will be RNAse free exactly for as long as the time you wait before you start grinding your tissues Do’h! I do use a fresh box of tips when doing RNA work, just because my other tip boses are always open. Keeping RNAse of your bench should not be exaggerated, just use common sense. RNAse does not fly through the walls of your tubes. Finaly, are you sure you have RNAse free isopropanol or ethanol? Haha, Scrutinized!
- grind ~30-50 mg of tissue
- add 300 μl of lysis solution and homogenize thoroughly, quickly
- incubate @ room temperature for 5 min (important step)
- add 100 μl of precipitation solution and mix by inverting the tubes gently.
- incubate 10 min @ 4◦C
- Spin at 4◦C for 10 min 11500 rpm.
- transfer 300 ul of supernatant to new tube (if there is still some material floating, centrifuge again).
- add 300 μl of isopropanol and mix by inverting
- spin 4 minutes and carefully pour of the sup
- wash pellet with 70% EtOH, air dry and resuspend pellet in 25 μl of RNAse-free water
- Add 3 μl 10X DNAse buffer and 2 μl of DNAseI
- Incubate 30 min at 37◦C (or whatever the specific enzyme requires you to do)
- precipitate: to the 30 μl of RNA, add 70 μl of RNAse free water, 50 μl of 7.5 M NH4Ac and 400 μl 95% EtOH and mix well.
- Spin 20 min at 4◦C, wash pellet with 70% EtOH, air dry RNA and resuspend in 20 μl of RNAse free water
solutionsLysis buffer (for 50 ml)
2 % SDS (5 ml 20% SDS)
68mM Sodium citrate FW: 294.10 (1.0 g)
132mM Citric acid FW: 210.14 (1.4 g)
1mM EDTA (0.1 ml 0.5 M EDTA) Precipitation solution (for 25 m)l
4M NaCl (20 ml 5 M NaCl)
16mM Sodium citrate (0.12 g)
32mM Citric acid (0.17 g) 7.5 M NH4Ac (FW: 77.0825) 28.9 g/50 ml
referenceOñate-Sánchez and Vicente-Carbajosa (2008). DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques.BMC Res Notes. Oct 20;1:93