David C H

Unless I am missing something, I don't see a problem with your setup.

Make sure your reagents are good. I assume you have used these reagents (minus the primers) successfully with something else. If not, test your reagents with template and primer that have had positive results in your hands or in your lab.

Beyond that, especially if you designed the primers yourself, make sure each primer set works by itself before combining multiple primer sets in the reaction.

You have not provided any details. If you want useful input, provide a very thorough description of what you did (reagent concentrations, cycling conditions, primer information).

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.

Enzyme storage buffers are often 50% glycerol with whatever buffer makes the enzyme happy. If you bought your taq, it should come with a manual or product information that lists the storage buffer (or you should be able to find this on their website). For example, Roche includes this with their taq: Enzyme storage buffer: 20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 0.1 M KCl, 0.5% Nonidet P40 (v/v), 0.5% Tween 20 (v/v), 50% glycerol (v/v), pH 8.0 (4°C)

What species or tissue to you intend to test? Some plants work well with any method. Others are much more difficult. Here are four options, in order of speed and ease, starting with the fastest and easiest.

1. Commercial kits generally use similar buffers and binding chemistries. The RNeasy kit from Qiagen is fast, easy, and works on many plants. For certain plants, RNeasy (and every other comercial kit I have tried) will fail to produce RNA suitable for RT-PCR.

2. Henderson, D. C. and J. Hammond (2013). "CKC: Isolation of Nucleic Acids from a Diversity of Plants Using CTAB and Silica Columns." Molecular Biotechnology 53(2): 109-117. This protocol uses organic extraction followed by column purification. It has produced RNA suitable for RT-PCR on every species I have tested (more than 80 species from over 25 families). However, yields from old leaves from some species are very low. A detailed protocol is included in the supplemental material downloadable from the publisher's website.

3. Li, R., R. Mock, et al. (2008). "A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens." Journal of Virological Methods 154(1-2): 48-55. Yields are higher than the previous protocol, especially with difficult samples but it also has a tendency to include more impurities. It has also been widely tested and in every case was acceptable for RT-PCR (because of impurities, some samples may be less suitable for Quantitative RT-PCR).

4. Chang, S., J. Puryear, et al. (1993). "A simple and efficient method for isolating RNA from pine trees." Plant Molecular Biology Reporter 11(2): 113-116. I have not tested this but from what I am told, this is likely to maximize yield, purity and reliability. However, it is much longer and more troublesome than the others.

There are papers (sorry, I don't have the references) that use an additional lysis buffer in combination with RNeasy or similar kits. These methods are almost as fast as RNeasy and were designed specifically for using RT-PCR on virus infections in plants. The yields are very low but good enough for RT-PCR. You should be able to find the references by searching for pcr detection of plant viruses.