Hi Murat,

low 260/280 ratios in RNA can be improved by purification via columns. I have very good  experience using the RNeasy Mini Kit by Qiagen with the RNA cleanup protocol. Nevertheless, if there is a degradation issue this of course will not be solved by the cleanup. When handling RNA always use RNase-free material (tips, tubes, water etc.).

Hi Murat,

Regarding the degradation pattern, in my experience sometimes you can see that high carbohydrate contamination looks similar to degradation in gel, so try to clean it a bit.

For seeds, where we also get high amounts of carbohydrates, we use high salt precipitation:

http://www.nippongene.com/english/tds/tds_high-salt-prep-sol-e1304.pdf

Although in this protocol they advise 0.5 vol, we use 1/4 vol isopropanol and 1/4 vol high salt solution, mix it well and leave it on ice for at least 2 hours. After that, remove the supernatant and rinse with EtOH 70% and finally dissolve in the appropiate volume of H2O.

Hope this works for you

Good luck!

Murat, you may also want to repeat the chloroform extraction multiple times, particularly if you see accumulated material at the interphase. I frequently did 3 subsequent chloroform extractions when isolating RNA using Trizol. Also, you do not describe the state of the leaf tissue that you are starting with, but I recommend flash-freezing of the tissue in liquid nitrogen prior to the extraction. Do not allow the tissue to thaw.

u might have phenol contamination in your RNA samples. repeat chloroform extraction step. u will see the difference.

Thank you all kind of your answers. 

Elisabeth, I have actually repeated chloroform extraction one more time, by adding equal volume of chloroform into aqueous phase after taken it into a new tube but results get worst. Maybe I have mistaken when I take supernatant from liquid liquid face. But I will try again for several repeat.

Leaf materials haven kept at -80 for 3 moths, I added 1 ml trizol directly on it and homogenised by bead microtube for 3 minutes. 

Also I just increase volume of chloroform (0,25 for 1 ml trizol)  then I got better 260/230 ratio (1.5).  But still agarose gel looks likes degraded. 

when you take supernatant from liquid liquid face, do it slowly and gentlely, and don't be too greedy to take too much. 

Degradation may be caused by RNAase  contaminated or  improper pretreatment of materials.

Hi Murat

The better and inexpensive way to improve the ratio of A260/230 is to do the second purification with PCI (phenol:chloroform:isoamylalcohol=25:24:1). The following is the protocol.

1. Dissolve RNA product with 200 ul DEPC-water, and then add the same volume PCI.

2. Vortex the sample in 4 oC for 5 min.

3. Centrifuge the sample in 4 oC for 10 min. at 12,000 rpm.

4. Transfer the supernatant to new tube, and add 1/10 volume 3M sodium acetate (pH 5.2) and 2.5 volume absolute ethanol. Mix well. If you want to increase the yield, you can incubate them in -20 oC for one hour or overnight.

5. Centrifuge the sample in 4 oC for 10 min. at 12,000 rpm.

6. Discard the supernatant and wash the pellet with 500 ul 70% ethanol.

7. Centrifuge the sample in 4 oC for 5 min. at 12,000 rpm.

8. Discard the supernatant and air dry the pellet.

9. Dissolve the pellet with 50 ul DEPC-water.

With this protocol, I think you can have good results.

I think I have solved the problem, Intense homogenisation might cause phenolic or carbohydrate contamination. I just squeezed leaf sample in liquid nitrogen by a pestle in 2 ml tube by hand instead of intense bead homogenisation. Then add 0,275 ml chloroform instead of 0, 2 ml after tizol, and repeat ethanol wash 3 time improved 260/230 ratio to 1.9 and  give very intact RNA band in agarose gel. 

Also PCI and repeated chloroform extraction worked well but yield drop too much (4 ug/ 40 ul), these would be very suitable for higher volume extraction with falcon tubes protocols.

Thanks allot for all lovely suggestion :)

Hello Murat,

Although I'm a bit late with the suggestions, I'd like to add that one likely reason for your degradation problem was the homogeneisation (as you show by using LN). 1ml [of Trizol] is a really big volume, so grinding won't be efficient, and cells can break inside the tissue due to pressure, releasing RNAses, before the trizol reaches them. Yields are likely to be low as well. Grinding frozen (dry ice)  tissue + beads will prove much more efficient, or alternatively your working LN protocol. As long as everything stays frozen (so you prevent RNAse action), you can grind to a fine powder and increase your yield. Then add the Trizol and immediately mix by vortexing to kill the RNases (you are showing that your problem was not equipment or buffer contamination, because now it's working).

As to the bad ratio, in my hands it heavily depends on the Trizol brand. I used the Ambion one and got OK ratios, but then tried the Sigma or the Thermo ones, and the ratios dropped. After the isopropanol, a second precipitation step with ammonium acetate (or LiCl, or you could also use standard sodium acetate) will give you 260/230 > 2. This will also help to get better yields, because phase-separation (further chloroform steps) always implies loss.

If you are interested in using columns for purification, you can use Qiagen or go for home-made alternatives (http://www.ncbi.nlm.nih.gov/pubmed/22260178). I have used this Logspin method and got the same yields and quality as with Qiagen, at a fraction of the price. If you work out the buffer concentrations, you can add the buffer directly to the sample (before the isopropanol) and circumvent the need for precipitation. Or if you want to do DNAse clean-up, you can adapt this protocol I developed for a desert plant.

Best,

Ruben

https://www.researchgate.net/file.PostFileLoader.html?id=552bb597d685cc8c4b8b4567&assetKey=AS%3A273755991937027%401442279922346

http://www.ncbi.nlm.nih.gov/pubmed/22260178