Hi everyone,
I am isolating RNA from pine cambium and am having problems getting clean pure RNA. If I use a miRvana kit I get around 10ng/ul RNA and 0,5 ng/ul DNA and by LiCl precipitation I get clean RNA samples but also higher contaminating DNA.
If I use DNase treatment (thermofisher AM1906) it only works on LiCl extracted RNA but does not efficiently remove DNA (from 5 to 1 ng/ul, after 2 hours doubling enzyme and buffer). With this conditions RNA starts to degrade.
I would like to know if Turbo DNase really works more efficiently and faster than the normal DNase and if someone has used both which is his/her experience.
Diego
I've found Turbo DNase to be more stable than DNase I've had to resuspend in water (cough cough Qiagen) and has a longer shelf life because I think they add glycerol + some other stuff to the solutions.
I've found that combining an on-column digestion + a post-isolation DNase digestion is the best way for me to get pure RNA without contamination DNA, but this is when I started using the Qiagen RNA extraction kit (new lab, new kits).
Hi Diego,
I'm really not familiar with plant cells or tissues, but as a general rule the best procedure to isolate clean RNA, if you don't need pre-mRNAs, is to do a cytoplasmic lysis with non-denaturing detergents (starting from Tween-20 or TritonX-100 up to stronger ones like deoxycholate, you can also use mixtures of the different detergents) and 5 mM MgCl2 in the lysis buffer: this will preserve the nuclear envelope intact and only minor amounts of gDNA will leak out. After a 10 min spin at 10000 g you can recover the supernatant containing most of the cytoplasmic RNA and add denaturing detergents, such as SDS, for further processing (PK digestion and phenol extraction) or dilute in lysis/binding buffers for column purification... Unfortunately the drawback of using kits is that you are not told what is in their solutions, so you are not given the possibility to choose how you want to process your samples...but you cab try to work around it, binding to a column does not depend too much on the volume or the dilution you load on it, but by the physical conditions (salt concentration, pH, hydrophobicity), so a 5-fold dilution in the provided buffer should work well enough...
Good luck
Thanks everyone for your answers!
I am attaching an image of my samples run on an agarose 1.2% gel for you to know how contaminated my samples are. From the gel it can be seen that the extraction method using LiCl is nice but also extracts some DNA.
Now, using DNase would be risky as it may degrade my RNA. However, I will consider using Turbo DNase if I do not have any other option.
Doing some research I have come across that acidic phenol can be used to remove DNA from RNA samples. At low phenol pHs like 4.5, DNA moves to the organic phase while RNA stays in the aqueous one. Does anybody have any experience on using acidic phenol for removing DNA from already purified RNA?
Thanks a lot for your help!
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