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Viral RNA extraction/isolation methods from plant tissue


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#1 sercanpazarlar

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Posted 27 February 2013 - 12:08 AM

Hello everyone

I am trying to find best viral RNA extraction method from plants. I tried a few methods "silica capture, Ethdyum bromide" and a kit RNAeasy. Could you please tell more methods (protocols) you clearly work?

Thank you

#2 David C H

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Posted 28 February 2013 - 01:08 PM

I may be able to offer some suggestions but more information is needed:

Are you trying to get viral RNA only? Or are you trying to get total RNA (plant and virus) from infected plant tissue?

What species of plant and what tissue are you interested in?

#3 sercanpazarlar

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Posted 01 March 2013 - 01:16 AM

I may be able to offer some suggestions but more information is needed:

Are you trying to get viral RNA only? Or are you trying to get total RNA (plant and virus) from infected plant tissue?

What species of plant and what tissue are you interested in?


Hi,
In fact I am triying to get viral RNA for reverse transcriptase PCR (to diagnose virus diseases in plants), but I did not know any method for only get viral RNA from any plant tissue. So I want to know the method that provide all RNA (plant, virus, viroid or others in) or total nucleic acids (plant, viruses, others RNA&DNA) then I can do cDNA before PCR.
I hope, I explain clearly
Thank you

#4 David C H

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Posted 01 March 2013 - 06:57 AM

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.

#5 sercanpazarlar

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Posted 04 March 2013 - 02:00 AM

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.


Hi,
Firstly thank you for your advices. I will try on grapevine tissues CTAB method and compare the results with silica capture, RNeasy, LiCl by nano drop. Than I can share.




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