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).

Turbo DNAse works just fine. I would use it.

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!

Hi Diego,

as I said, plants are not my field, but looking at your gel, I'm not sure you are dealing with gDNA contamination. Is your marker a 100 bp, with the upper two bands at 1.5 and 3 kb? Then your upper band is around 4 kb and the middle one around 1.5, which would fit with the two rRNA species... Estimating RNA size with a DNA ladder (that is double stranded) is not the best.... I would wonder what is the lowest band, possibly around 1 kb, but plants have also the chloroplastic rRNA, so there is often heterogeneity.

gDNA contamination could be the small band in samples 4-6 just below the wells... it's a 1.2% gel and gDNA is million bases per chromosome, it wouldn't migrate that far into your gel...

A few more general comments:

LiCl precipitation can be done in two ways, adding 0.8 M LiCl and then 2.5 vol of EtOH, but this is not selective for RNA! Alternatively, directly in 2.5 M LiCl (adding to your RNA solution 1 vol of an ice-cold 5 M LiCl solution and mixing very thoroughly by inversion and flipping the tube, not by vortexing!) on ice or at -20 °C from a minimum of 2 hrs to O/N (if you have little material, but the approach requires a minimal concentration of 50-100 ng/ul of RNA). Then spin 30 min at 4 °C and very carefully remove the super because the pellet is absolutely invisible! As soon as you add some 70 % EtOH to wash, the pellet will whiten and become visible... This method is VERY selective for RNA, almost all gDNA, nucleotides and small fragments from degradation (