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Extracting total RNA from woody plant roots

RNA shrub roots

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#1 cinnamon wattle

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Posted 01 April 2013 - 08:11 PM

Hi,

Does anyone have experience extracting total RNA from woody plant roots? I'm attempting to extract total RNA from different Acacia spp. Initially, I tried Bioline's Isolate Plant RNA kit and then tried TriSure to no success. My last attempts were using 65 C CTAB incubation, a chloroform addition, and LiCl precipitation after the phenol/chloroform cleaning steps. This produced a white pellet, but no detectable RNA (by nanodrop or on a 1.5% agarose gel). If I cleaned the pellet a second time w/phenol/chloroform and precipitated with 3M sodium acetate, this did not improve my result. All the solutions were made using DEPC-treated water, and other materials were baked at 200 C to prevent RNase contamination. However, I did not buffer my phenol (it is quite acidic), having read that acidic phenol preferentially dissolved RNA. Could this be the problem, or does anyone have any other suggestions? Ta!

#2 David C H

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Posted 02 April 2013 - 08:17 AM

How acidic is your phenol? Trizol (phenol/chloroform, guanidine salts, and something else) is buffered to acidic pH. The low pH causes most of the DNA to precipitate in the interphase, leaving RNA in the supernatant.

If you think the phenol is making the solution too acidic, do the organic extraction without the phenol (chloroform plus lysis buffer). If this works, you will also have some DNA in your final product (usually 10-25%; RNA kits still yield 5-15% DNA, depending on the organism. Kits with on-column digestion of DNA still have 0.5-2% DNA).

It is notoriously difficult to get nucleic acid out of roots. The white pellet you are getting is probably some type of polysaccharide.

Some suggestions:

Use half of the tissue that the protocol recommends (if it is 100mg, use 50mg or less).

Use the youngest, softest tissue possible.

Use a FastPrep or similar instrument with 6.35mm (1/4 inch) steel beads in Toughtubes (MoBio) to disrupt tissue (this usually disrupts tissue more thoroughly than mortar and pestle, increasing the yield).

Use a buffer with CTAB and PVP (or PVPP).

Instead of 65deg, incubate at 56deg or skip the incubation -- the high heat helps CTAB detergent break up cell membranes, but it also messes with some polysaccharides and causes them to be more of a problem. RNases are usually still moderately active in most CTAB buffers; they are less active at 65deg, so do not incubate for more than five minutes at lower temperatures. With good disruption (FastPrep plus beads), you may be able to skip the 65deg step altogether.

I can suggest some papers/protocols if needed.

How much RNA do you need? If you only need to to RT-PCR, doing the isolation with a CTAB/PVP buffer and then adding a fraction of that to your Bioline kit may be sufficient. The yield will be very low, but enough for RT-PCR.

Edited by David C H, 02 April 2013 - 08:24 AM.


#3 cinnamon wattle

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Posted 04 April 2013 - 08:26 PM

Hi David,

Thanks very much for the prompt and detailed reply. I want the RNA to construct cDNA that will then be used in PCR. Arguably, then, I only need a few micrograms.

I've now tried using CTAB/PVP with only chloroform:IAA extraction without success. So if you can recommend some papers/protocols, it would be fantastic, particularly if the protocol has all the steps detailed rather than assumed. (I tried using one from Chang et al, '93, Plant Mol Bio Rep, but it seemed so succinct that steps were omitted).

Would adding an RNAse inhibitor (from the cDNA kit) to the CTAB step help preserve any RNA, do you think?

Thanks again.
V





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