Comments on: 10 Tips for the Isolation of High Quality RNA from Soil http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/ Tue, 13 Dec 2011 00:32:47 +0000 http://wordpress.org/?v=2.9.1 hourly 1 By: Suzanne Kennedy http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/comment-page-1/#comment-11072 Suzanne Kennedy Fri, 23 Sep 2011 16:23:33 +0000 http://www.mobio.com/blog/?p=882#comment-11072 Hi Katerine, Sometimes I see this also- this is at step 11-12, right? I just mix it up before I centrifuge the sample. The key is that the pellet after the 30 minute spin is flat and translucent and it is usually brown. It shouldn't be salty or thick or crystal like. What does the pellet look like? Change the incubation step to room temperature instead of 4C. The phases are not as much a problem as the salt. A lot of marine sediments are very salty so this might be why you are having a problem, if you used -20C incubation. I will email you directly also so we can continue the discussion off line. best, Suzanne Hi Katerine,
Sometimes I see this also- this is at step 11-12, right? I just mix it up before I centrifuge the sample. The key is that the pellet after the 30 minute spin is flat and translucent and it is usually brown. It shouldn’t be salty or thick or crystal like. What does the pellet look like? Change the incubation step to room temperature instead of 4C. The phases are not as much a problem as the salt.
A lot of marine sediments are very salty so this might be why you are having a problem, if you used -20C incubation.
I will email you directly also so we can continue the discussion off line.
best,
Suzanne

]]> By: Katherine http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/comment-page-1/#comment-11068 Katherine Thu, 22 Sep 2011 20:36:18 +0000 http://www.mobio.com/blog/?p=882#comment-11068 Hello, I have been using this kit to extract RNA from marine sediments and while I was looking at troubleshooting guide I found this blog. It is quite interesting all the tips that you have mentioned and they have been very useful. But I have the same problem as John, in the precipitation step I always see thwo phases and it is not phenol that has been carrying over. I think the formation of the two phases is a problem because I haven' t been able to extract RNA and if there is any, it won't precipitate. The question is how do I stop the formation of these two phases? how can I avoid this? Hello,
I have been using this kit to extract RNA from marine sediments and while I was looking at troubleshooting guide I found this blog. It is quite interesting all the tips that you have mentioned and they have been very useful. But I have the same problem as John, in the precipitation step I always see thwo phases and it is not phenol that has been carrying over. I think the formation of the two phases is a problem because I haven’ t been able to extract RNA and if there is any, it won’t precipitate. The question is how do I stop the formation of these two phases? how can I avoid this?

]]> By: Suzanne Kennedy http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/comment-page-1/#comment-6678 Suzanne Kennedy Thu, 09 Sep 2010 18:01:54 +0000 http://www.mobio.com/blog/?p=882#comment-6678 Hi John, I think I just emailed you directly too... So this next try was with new phenol? Normally I do not see phases after the centrifugation. All you want after this step is the pellet. Did you have a pellet underneath the liquid? I wonder if the phases you see are from carry over of some of the phenol after the extraction. After extraction in the phenol, with our soils, there is only an upper aqueous and the phenol and soil form a mass so we don't get carry over of phenol. What is your soil like? What is the texture? You can email me directly also. Suzanne Hi John,
I think I just emailed you directly too…
So this next try was with new phenol? Normally I do not see phases after the centrifugation. All you want after this step is the pellet. Did you have a pellet underneath the liquid?
I wonder if the phases you see are from carry over of some of the phenol after the extraction. After extraction in the phenol, with our soils, there is only an upper aqueous and the phenol and soil form a mass so we don’t get carry over of phenol.
What is your soil like? What is the texture?
You can email me directly also.
Suzanne

]]> By: john http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/comment-page-1/#comment-6650 john Wed, 08 Sep 2010 23:29:34 +0000 http://www.mobio.com/blog/?p=882#comment-6650 Hello Suzanne: I tried the RNA powersoil kit for three times and got nothing from it. I am wondering what is the problem. in today's extraction, I strictly follow the protocol, at step 12, after the incubation at room temperature, I observed a very clear two-phase separation (same as I observed for the last two try), the lower phase is about 2 ml, with very light or almost no color, DO you have any idea what it is? It smells like phenol but I did not transfer any lower phase at step 8 and I did not observe this at step 9/10/11. I called your technical people and Nik said that he did not see this before. I am wondering what is wrong? This almost make me mad. Please help me, thanks Hello Suzanne:
I tried the RNA powersoil kit for three times and got nothing from it. I am wondering what is the problem.
in today’s extraction, I strictly follow the protocol, at step 12, after the incubation at room temperature, I observed a very clear two-phase separation (same as I observed for the last two try), the lower phase is about 2 ml, with very light or almost no color, DO you have any idea what it is? It smells like phenol but I did not transfer any lower phase at step 8 and I did not observe this at step 9/10/11. I called your technical people and Nik said that he did not see this before. I am wondering what is wrong? This almost make me mad. Please help me, thanks

]]> By: john http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/comment-page-1/#comment-6610 john Tue, 07 Sep 2010 13:53:02 +0000 http://www.mobio.com/blog/?p=882#comment-6610 Hi Suzanne: Thank you for the suggestion, I mean 30+30 sec, that is a first 30 sec followed by another 30 sec. and then repeat another one cycle. I will try you suggestions (change to new batch of Phenol) and let you know what happens. all the best JOhn Hi Suzanne:
Thank you for the suggestion, I mean 30+30 sec, that is a first 30 sec followed by another 30 sec. and then repeat another one cycle.
I will try you suggestions (change to new batch of Phenol) and let you know what happens.
all the best

JOhn

]]> By: Suzanne Kennedy http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/comment-page-1/#comment-6605 Suzanne Kennedy Tue, 07 Sep 2010 06:18:34 +0000 http://www.mobio.com/blog/?p=882#comment-6605 Hi John, Thanks for reading the blog and your comments. For your first question, do you mean after elution from the gravity column why not use a spin filter? I suppose it would be possible to do that. It just adds cost to include another column. Since the elution is in 1 ml, you would need to add 1 ml of an RNA binding solution and 1 ml of ethanol. It would require 5 x 600 ul centrifugations to load it all in. Sometimes the RNA pellet is very difficult to see because it is clean. What we do is orient the tubes in the centrifuge so we know exactly where the pellet will be. When I decant the superntant after the centrifugation at the end, I look at that spot. It may not always look like a pellet. It can sometimes look like a little tiny spec on the tube. Just resuspend it with water. For samples with very low RNA content, I use 25 ul for resuspension so I can quantitate it easier. The RNA pellet is probably there so just resuspend the spot where it should be. How much DNA do you get from this same soil if you do a PowerSoil DNA prep? The phenol should not be red, yellow, or pink. Here is more info on the phenol. Did you buy one of the brands we recommend in our manual? Our phenol is always clear. Did you buy some fresh phenol or is this very old. I would highly recommend getting a new bottle. We use the Amresco phenol (mentioned in the manual) in our lab. <em>Phenol and phenol:chloroform:isoamyl alcohol are subject to oxidation reactions that cause them to become yellow or pink colored, which serves as an indicator that the phenol is NOT useable for RNA extraction. Using colored phenol or colored phenol:chloroform:isoamyl alcohol will result in quality compromised RNA. Prior to each use, a sample of the phenol:chloroform:isoamyl alcohol should be placed in a clear container and its clarity determined. Oxidized phenol damages nucleic acids.</em> For the FastPrep, you are doing 30 x 30 seconds? This might be too much. The phenol is going to lyse the oocyst. I don't think the oocyst can survive phenol. I would try the prep again but using our protocol with the 10 minutes vortexing in phenol. This likely works just fine. You may want to cut back the FastPrep settings to only 3-5 cycles of 30 seconds. Too much power on the FastPrep will heat the sample and heating in combination with the phenol will degrade the RNA. I think the bead type we use for the RNA PowerSoil is ok for the FastPrep but maybe you are doing too many cycles. We do have some glass bead tubes in 15 ml size you could try out. It might be a better idea to buy some of the glass beads in bulk (0.5 mm glass beads) and then add 1-2 grams to the RNA PowerSoil bead tube to give it harder beads for the FastPrep. Then, you may be able to cut back to only 2-3 cycles at 30 seconds. The reduced cycles will reduce the heating and be better for the RNA. The vortex does not generate near the amount of heat that high powered bead beating does. And the last question on BME, yes, I have added it to soil preps and it works fine. It won't be a problem with the prep. Add 10 ul per 1 ml of bead solution. It can protect, although, in the phenol, all the RNases will be killed. Phenol will permanently denature all the proteins. But if you were worried about the initial 5 minute vortex before the phenol is added, you could add some BME and it would destroy the RNases present in the soil. Hi John,
Thanks for reading the blog and your comments. For your first question, do you mean after elution from the gravity column why not use a spin filter? I suppose it would be possible to do that. It just adds cost to include another column. Since the elution is in 1 ml, you would need to add 1 ml of an RNA binding solution and 1 ml of ethanol. It would require 5 x 600 ul centrifugations to load it all in.

Sometimes the RNA pellet is very difficult to see because it is clean. What we do is orient the tubes in the centrifuge so we know exactly where the pellet will be. When I decant the superntant after the centrifugation at the end, I look at that spot. It may not always look like a pellet. It can sometimes look like a little tiny spec on the tube. Just resuspend it with water. For samples with very low RNA content, I use 25 ul for resuspension so I can quantitate it easier.
The RNA pellet is probably there so just resuspend the spot where it should be.

How much DNA do you get from this same soil if you do a PowerSoil DNA prep?

The phenol should not be red, yellow, or pink. Here is more info on the phenol. Did you buy one of the brands we recommend in our manual? Our phenol is always clear. Did you buy some fresh phenol or is this very old. I would highly recommend getting a new bottle. We use the Amresco phenol (mentioned in the manual) in our lab.

Phenol and phenol:chloroform:isoamyl alcohol are subject to oxidation reactions that cause them to become yellow or pink colored, which serves as an indicator that the phenol is NOT useable for RNA extraction. Using colored phenol or colored phenol:chloroform:isoamyl alcohol will result in quality compromised RNA. Prior to each use, a sample of the phenol:chloroform:isoamyl alcohol should be placed in a clear container and its clarity determined. Oxidized phenol damages nucleic acids.

For the FastPrep, you are doing 30 x 30 seconds? This might be too much. The phenol is going to lyse the oocyst. I don’t think the oocyst can survive phenol. I would try the prep again but using our protocol with the 10 minutes vortexing in phenol. This likely works just fine. You may want to cut back the FastPrep settings to only 3-5 cycles of 30 seconds. Too much power on the FastPrep will heat the sample and heating in combination with the phenol will degrade the RNA.

I think the bead type we use for the RNA PowerSoil is ok for the FastPrep but maybe you are doing too many cycles.
We do have some glass bead tubes in 15 ml size you could try out. It might be a better idea to buy some of the glass beads in bulk (0.5 mm glass beads) and then add 1-2 grams to the RNA PowerSoil bead tube to give it harder beads for the FastPrep. Then, you may be able to cut back to only 2-3 cycles at 30 seconds. The reduced cycles will reduce the heating and be better for the RNA. The vortex does not generate near the amount of heat that high powered bead beating does.

And the last question on BME, yes, I have added it to soil preps and it works fine. It won’t be a problem with the prep. Add 10 ul per 1 ml of bead solution.
It can protect, although, in the phenol, all the RNases will be killed. Phenol will permanently denature all the proteins. But if you were worried about the initial 5 minute vortex before the phenol is added, you could add some BME and it would destroy the RNases present in the soil.

]]> By: john http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/comment-page-1/#comment-6597 john Mon, 06 Sep 2010 23:13:19 +0000 http://www.mobio.com/blog/?p=882#comment-6597 another question for Suzanne: Can I add BME to the lysis buffer in the kit to completely remove RNase from the soil sample? Thank you another question for Suzanne:

Can I add BME to the lysis buffer in the kit to completely remove RNase from the soil sample? Thank you

]]> By: john http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/comment-page-1/#comment-6596 john Mon, 06 Sep 2010 23:10:59 +0000 http://www.mobio.com/blog/?p=882#comment-6596 Hello, Suzanne: this is a very good blog, i learned a lot new things, thank you. I am now using RNA Powersoil kit to extract RNA from different soil, I have one question: why not use spin filter in the final step instead of the 2 ml tube? I can not see the pellet of RNA each time, which makes me wonder whether there is any RNA. I was not succesful for the last two tries. I called the Tech Support and was told that in the final step just be very careful, I was VERY careful but still cannot see anything. another question: the Phenol-Cloroform-isoethanol is a little yellow, do you think this may cause some problem? if yes, what will be the effect? I do not think the vortex is strong enough to break the cell wall of my oocyst (from which I will extract the RNA), So I use the FASTPREP at set 5.5 or set 6 for 30+30, then 30+30 sec, Do you think fastprep is too strong for RNA extraction from soil? Do you think this will affect the integrity of RNA? From your another blog I learned that the bead type in the Powersoil RNA kit is designed for vortex, So if I use this type of tube in the fastprep, will it not work? Thank you a lot for any suggestion on the extraction of RNA from Soil. Best regards from Oklahama John Hello, Suzanne:
this is a very good blog, i learned a lot new things, thank you.
I am now using RNA Powersoil kit to extract RNA from different soil, I have one question: why not use spin filter in the final step instead of the 2 ml tube? I can not see the pellet of RNA each time, which makes me wonder whether there is any RNA. I was not succesful for the last two tries. I called the Tech Support and was told that in the final step just be very careful, I was VERY careful but still cannot see anything.
another question: the Phenol-Cloroform-isoethanol is a little yellow, do you think this may cause some problem? if yes, what will be the effect?
I do not think the vortex is strong enough to break the cell wall of my oocyst (from which I will extract the RNA), So I use the FASTPREP at set 5.5 or set 6 for 30+30, then 30+30 sec, Do you think fastprep is too strong for RNA extraction from soil? Do you think this will affect the integrity of RNA?
From your another blog I learned that the bead type in the Powersoil RNA kit is designed for vortex, So if I use this type of tube in the fastprep, will it not work?
Thank you a lot for any suggestion on the extraction of RNA from Soil.

Best regards from Oklahama

John

]]> By: Suzanne Kennedy http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/comment-page-1/#comment-6406 Suzanne Kennedy Sun, 29 Aug 2010 00:08:49 +0000 http://www.mobio.com/blog/?p=882#comment-6406 Hi Virginia- I'll fix it- sorry about that! Suzanne Hi Virginia- I’ll fix it- sorry about that!
Suzanne

]]> By: Virginia http://www.mobio.com/blog/2010/03/23/10-tips-for-the-isolation-of-high-quality-rna-from-soil/comment-page-1/#comment-6366 Virginia Wed, 25 Aug 2010 11:05:04 +0000 http://www.mobio.com/blog/?p=882#comment-6366 Hi there. The link in the soil storage section for "More information including data can be found here." seems to be broken - just a very small jpg image of what could be the document of interest. It would be great to get this info. Thanks so much! Hi there. The link in the soil storage section for “More information including data can be found here.” seems to be broken – just a very small jpg image of what could be the document of interest. It would be great to get this info. Thanks so much!

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