A Gel of a Problem: running miRNA without acrylamide
May 30, 2013 2:40 PM Subscribe
Dear MetaFilter, I am new to this forum and this is my first post, hi!
I am in a bit of a pickle about a work project, and hope that I may be able to get some help by some fellow sciencey people. The science forums are rarely read and badly out of date, or I would have posted at one of those.
I have a deadline for a miRNA PCR experiment, and I have already run the cDNA and PCR for the miRNA. All I have to do is run the miRNA on a gel.
The problem: We don't have acrylamide! And neither does our neighboring lab. We only have Agarose with which to make gels, which is only for large fragments, not small miRNA fragments of 20-40 bp.
The question: As the percentage of agarose in the gel goes higher, the smaller the size of the base pairs are that can be measured. Could I increase this percentage enough to work for miRNA?
Out of respect for the community that doesn't like line returns, I have entered the specifics of this post in the Extended Explanation box, because to me, this post reads like a jumble of nonsense without some kind of organization.
I have a deadline for a miRNA PCR experiment, and I have already run the cDNA and PCR for the miRNA. All I have to do is run the miRNA on a gel.
The problem: We don't have acrylamide! And neither does our neighboring lab. We only have Agarose with which to make gels, which is only for large fragments, not small miRNA fragments of 20-40 bp.
The question: As the percentage of agarose in the gel goes higher, the smaller the size of the base pairs are that can be measured. According to the first chart in this link:
(sorry, but MF won't let me paste this in the "link" box)
http://www.idtdna.com/pages/decoded/decoded-articles/pipet-tips/decoded/2011/06/17/running-agarose-and-polyacrylamide-gels
would it be feasible to follow the increasing trend of agarose up to, say, 2.0%, to attempt to measure bps 50 and below? Or is that something that just can't be done because of some special properties of acrylamide that need to be used with small fragments? I can't find an answer on the regular internets. I do have extra miRNA cDNA in the freezer if the experiment bombs the first time.
I would greatly appreciate any advice or shared experiences anyone has to offer. in other words, HAAAAALP! Thank you very much, and have a mice day!
P.S., I apologize to those of you who mentioned in a post that they do not like the separation of a post into paragraphs or line breaks http://metatalk.metafilter.com/20546/Just-say-no-to-multiple-paragraphs, but I did search the MeTa and the FAQ under several different terms and did not find any information for or against making paragraphs, line returns, line breaks etc. I previewed this post without paragraphs, and it was a mess and difficult to read.
I have a deadline for a miRNA PCR experiment, and I have already run the cDNA and PCR for the miRNA. All I have to do is run the miRNA on a gel.
The problem: We don't have acrylamide! And neither does our neighboring lab. We only have Agarose with which to make gels, which is only for large fragments, not small miRNA fragments of 20-40 bp.
The question: As the percentage of agarose in the gel goes higher, the smaller the size of the base pairs are that can be measured. According to the first chart in this link:
(sorry, but MF won't let me paste this in the "link" box)
http://www.idtdna.com/pages/decoded/decoded-articles/pipet-tips/decoded/2011/06/17/running-agarose-and-polyacrylamide-gels
would it be feasible to follow the increasing trend of agarose up to, say, 2.0%, to attempt to measure bps 50 and below? Or is that something that just can't be done because of some special properties of acrylamide that need to be used with small fragments? I can't find an answer on the regular internets. I do have extra miRNA cDNA in the freezer if the experiment bombs the first time.
I would greatly appreciate any advice or shared experiences anyone has to offer. in other words, HAAAAALP! Thank you very much, and have a mice day!
P.S., I apologize to those of you who mentioned in a post that they do not like the separation of a post into paragraphs or line breaks http://metatalk.metafilter.com/20546/Just-say-no-to-multiple-paragraphs, but I did search the MeTa and the FAQ under several different terms and did not find any information for or against making paragraphs, line returns, line breaks etc. I previewed this post without paragraphs, and it was a mess and difficult to read.
Not sure how helpful this will be, but I used to (in my lab days), run my PCR products on MetaPhor Agarose, which was higher resolution than our standard stuff.
We routinely used this to size DNA fragments where the ∆ from our standard would be < 50bp (though total fragment size was more like 500bp)
Not cheap stuff, but may be worth trying if your deadline is in days rather than hours...
posted by weaponsgradecarp at 3:02 PM on May 30, 2013 [1 favorite]
We routinely used this to size DNA fragments where the ∆ from our standard would be < 50bp (though total fragment size was more like 500bp)
Not cheap stuff, but may be worth trying if your deadline is in days rather than hours...
posted by weaponsgradecarp at 3:02 PM on May 30, 2013 [1 favorite]
from here, found via thread at http://network.nature.com/groups/natureprotocols/forum/topics/10621:
Most agarose gels are made between 0.7% and 2%. ... A 2% gel will show good resolution for small fragments (0.2–1 kb). Some people go as high as 3% for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. ... High percentage gels are often brittle and do not set evenly.
This page suggests to use 2.5% when you've got less than 75bp.
posted by neda at 3:12 PM on May 30, 2013 [1 favorite]
Most agarose gels are made between 0.7% and 2%. ... A 2% gel will show good resolution for small fragments (0.2–1 kb). Some people go as high as 3% for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. ... High percentage gels are often brittle and do not set evenly.
This page suggests to use 2.5% when you've got less than 75bp.
posted by neda at 3:12 PM on May 30, 2013 [1 favorite]
Response by poster: Thank you SO very much for all of your answers! They are so very helpful, I'm running the gel today and hoping to get results tomorrow.
Yes, the agarose and everything is RNAse free, at least it says so on the box. It is Fisher Molecular Biology Grade. Would I use the same percent of EtBr as a usual gel?
The links were so helpful; it appears that I'm not that great with my search terms.
I will try a high 2.5% gel, and run it very slowly as advised. I have nowhere to be and love the lab.
And thank you for the MetaPhor info, I'll ask my PI on our next order.
Again, thank you enormously for your help! Metafilter is even better than my wildest dreams. Ahem...I am a bit excited about getting such helpful answers.
posted by Arachnophile at 3:17 PM on May 30, 2013 [4 favorites]
Yes, the agarose and everything is RNAse free, at least it says so on the box. It is Fisher Molecular Biology Grade. Would I use the same percent of EtBr as a usual gel?
The links were so helpful; it appears that I'm not that great with my search terms.
I will try a high 2.5% gel, and run it very slowly as advised. I have nowhere to be and love the lab.
And thank you for the MetaPhor info, I'll ask my PI on our next order.
Again, thank you enormously for your help! Metafilter is even better than my wildest dreams. Ahem...I am a bit excited about getting such helpful answers.
posted by Arachnophile at 3:17 PM on May 30, 2013 [4 favorites]
You have three problems.
Your first problem is resolution. I regularly run gels to check qRT-PCR products (75-150bp) and very small PCR products using NuSieve 3:1 agarose, at 4%. I get pretty good resolution with that. Also not cheap, but effective. I have 10bp and 20bp ladders from Thermo and can very clearly resolve even the lowest bands with this setup. DNA not RNA, obviously, but there are similarly small RNA ladders (e.g. the Ambion Decade one.) I've also run standard 2% or 2.5% agarose gels on similarly-sized things; I would not rely on that working super-well for stuff below 50bp. (I mean, you can still see bands, but my smallest bands - even on my ladders - are blurrier and it would be harder to accurately differentiate the sizes of, say, a 35nt fragment vs. a 30nt fragment.) You have to be much more careful when pouring high percent agarose gels - Lonza's NuSieve instructions have served me well there.
Oh, and I run with TBE (and not TAE) when running gels for very small things. It can take higher voltages/temperatures better, and I get better resolution for small stuff with it. That goes for DNA and RNA.
Your second problem is secondary structure. Compared to non-plasmid DNA, RNA is way more likely to form all kinds of secondary structures that will make samples run unpredictably in non-denaturing gels - like a standard agarose gel. If you need accurate sizes, you need to run a denaturing gel, and formaldehyde is the standard answer for that (in agarose gels.) (Urea is used for denaturing nucleic acid polyacrylamide gels.)
The third problem is visualization. What do you usually do to visualize your RNA on polyacrylamide gels? If you silver stain, for example, I would be very dubious about that working on agarose. Ethidium bromide - which depends on nucleic acid intercalation - isn't going to work all that well in a denaturing RNA gel. (With small fragments, the differences in secondary structure may make it pretty unpredictable even in a native agarose gel.) Even in a native gel, smaller samples can yield fainter EtBr signals for obvious reasons. The kind of gel you run will affect both your visualization method and the sensitivity you can achieve with that method.
posted by ubersturm at 5:10 PM on May 30, 2013 [2 favorites]
Your first problem is resolution. I regularly run gels to check qRT-PCR products (75-150bp) and very small PCR products using NuSieve 3:1 agarose, at 4%. I get pretty good resolution with that. Also not cheap, but effective. I have 10bp and 20bp ladders from Thermo and can very clearly resolve even the lowest bands with this setup. DNA not RNA, obviously, but there are similarly small RNA ladders (e.g. the Ambion Decade one.) I've also run standard 2% or 2.5% agarose gels on similarly-sized things; I would not rely on that working super-well for stuff below 50bp. (I mean, you can still see bands, but my smallest bands - even on my ladders - are blurrier and it would be harder to accurately differentiate the sizes of, say, a 35nt fragment vs. a 30nt fragment.) You have to be much more careful when pouring high percent agarose gels - Lonza's NuSieve instructions have served me well there.
Oh, and I run with TBE (and not TAE) when running gels for very small things. It can take higher voltages/temperatures better, and I get better resolution for small stuff with it. That goes for DNA and RNA.
Your second problem is secondary structure. Compared to non-plasmid DNA, RNA is way more likely to form all kinds of secondary structures that will make samples run unpredictably in non-denaturing gels - like a standard agarose gel. If you need accurate sizes, you need to run a denaturing gel, and formaldehyde is the standard answer for that (in agarose gels.) (Urea is used for denaturing nucleic acid polyacrylamide gels.)
The third problem is visualization. What do you usually do to visualize your RNA on polyacrylamide gels? If you silver stain, for example, I would be very dubious about that working on agarose. Ethidium bromide - which depends on nucleic acid intercalation - isn't going to work all that well in a denaturing RNA gel. (With small fragments, the differences in secondary structure may make it pretty unpredictable even in a native agarose gel.) Even in a native gel, smaller samples can yield fainter EtBr signals for obvious reasons. The kind of gel you run will affect both your visualization method and the sensitivity you can achieve with that method.
posted by ubersturm at 5:10 PM on May 30, 2013 [2 favorites]
Ubersturm has excellent advice that supercedes mine; all I would say is to use a good SYBR stain, and soak for 1-2h if you use agarose.
e.g.
https://products.invitrogen.com/ivgn/product/S7564
posted by lalochezia at 5:40 PM on May 30, 2013
e.g.
https://products.invitrogen.com/ivgn/product/S7564
posted by lalochezia at 5:40 PM on May 30, 2013
Response by poster: Thank you for the advice. Yes, I am concerned about the resolution, I guess I'll find out soon!
I ended up making a 3% gel, which luckily and with a lot of patience, managed to get the comb out and put it in place without any cracks.
I definitely plan on using the denaturing gel in the future, my PI said I should order the urea polyacrylamide at 2.5% or 7.5%. Which do you recommend?
I know that I should have been more prepared, but we haven't done any miRNA stuff in a few years, and somehow we all thought that we were stocked with acrylamide. Which brings me to my first mistake; not physically checking my supplies before starting. Bad scientist!
We use EtBr for our agarose, which might be ok this time because I don't have a denaturing gel.
But this is a first run, so if I get even just fuzzy bands, that will show that we're on the right track, and make my boss happy. Then I'll order all of the right supplies for the next run. I have a lot of extra cDNA so I'll just have to repeat the PCR and try again, this time being more prepared.
It's running now! http://500px.com/photo/36144502
posted by Arachnophile at 5:58 PM on May 30, 2013 [2 favorites]
I ended up making a 3% gel, which luckily and with a lot of patience, managed to get the comb out and put it in place without any cracks.
I definitely plan on using the denaturing gel in the future, my PI said I should order the urea polyacrylamide at 2.5% or 7.5%. Which do you recommend?
I know that I should have been more prepared, but we haven't done any miRNA stuff in a few years, and somehow we all thought that we were stocked with acrylamide. Which brings me to my first mistake; not physically checking my supplies before starting. Bad scientist!
We use EtBr for our agarose, which might be ok this time because I don't have a denaturing gel.
But this is a first run, so if I get even just fuzzy bands, that will show that we're on the right track, and make my boss happy. Then I'll order all of the right supplies for the next run. I have a lot of extra cDNA so I'll just have to repeat the PCR and try again, this time being more prepared.
It's running now! http://500px.com/photo/36144502
posted by Arachnophile at 5:58 PM on May 30, 2013 [2 favorites]
I've routinely run non-denaturing 3% and even up to 4% *gasp* agarose gels for small stuff. But in the name of all that is holy ditch that evil EtBr!! Shiz gon' kill ya. We use GelRed (Biotium) and image w/ a UV transilluminator. There are some SYBR based products that are way less toxic too. Have you tried the Biotechniques Molecular Biology forum? Answers might not be immediate but sometimes are and I've always found that to be a great source of knowledge for real-life (real-lab?) questions like this. I'm pretty sure it was my best mentor during my PhD... :|
posted by SinAesthetic at 6:42 AM on May 31, 2013
posted by SinAesthetic at 6:42 AM on May 31, 2013
...we haven't done any miRNA stuff in a few years, and somehow we all thought that we were stocked with acrylamide.
That may be for the best - acrylamide doesn't last forever.
I definitely plan on using the denaturing gel in the future, my PI said I should order the urea polyacrylamide at 2.5% or 7.5%. Which do you recommend?
I'm not clear on what you mean by "urea polyacrylamide at 2.5% or 7.5%" - do you mean precast gels? I admit that I don't do precast gels, since pouring your own lets you tweak gels if it turns out your sample needs different conditions. (Actually, the instructions for Invitrogen's Novex TBE-urea ones confuse me: one is usually advised to avoid storing urea gels at 4ºC due to precipitation, so I am susprised they say their gels can last weeks at that temperature. The longest I've ever let a urea-TBE gel sit is overnight at room temperature, for polymerization.)
Regardless of whether you buy precast gels or make them yourself, though, I can't imagine why you would want 2.5% acrylamide (?!) for 20-40nt miRNA, unless there's some technique downstream that explicitly requires ridiculously low acrylamide amounts. I would think that 6% (a pretty frequent polyacrylamide concentration for nucleic acids) would really be the absolute minimum, and you'd really want to go rather higher if your miRNA is in the 20-40nt range (which is only oligo size!) Probably closer to 15%, really. Just as higher percentages of agarose are used for resolving small things, higher percentages of acrylamide are used for resolving small things. Most manufacturers of electrophoresis stuff have guides that can help you choose the right percentage and so on, and which often include information on less-used techniques, the science behind electrophoresis, and so on. This is one I keep a pdf of due to the sheer number of techniques discussed, but National Diagnostics has a bunch of DNA/RNA-focused electrophoresis info on their site, there's a good chapter in a molecular bio book, etc.
But in the name of all that is holy ditch that evil EtBr!! Shiz gon' kill ya.
Meh. The problem with EtBr is mutagenicity, not toxicity. A lot of the newer alternatives are possibly less mutagenic (when there is data available), but have higher acute toxicity - and are often dissolved in DMSO, potentially making it easier for them to get into cells. (A post by a molecular biologist discussing this.) Anything that binds DNA has at least some potential to be problematic. Furthermore, any number of other things discussed here - formaldehyde, acrylamide, arguably the UV used to view EtBr gels - are more dangerous - but none of them should be a problem as long as you work safely, with proper protective equipment. Ethidium bromide is cheap and effective, and though there may be better options if you can afford them (for sensitivity and avoiding nucleic acid UV exposure, not just safety), it is highly unlikely to kill you.
posted by ubersturm at 1:49 PM on May 31, 2013 [1 favorite]
That may be for the best - acrylamide doesn't last forever.
I definitely plan on using the denaturing gel in the future, my PI said I should order the urea polyacrylamide at 2.5% or 7.5%. Which do you recommend?
I'm not clear on what you mean by "urea polyacrylamide at 2.5% or 7.5%" - do you mean precast gels? I admit that I don't do precast gels, since pouring your own lets you tweak gels if it turns out your sample needs different conditions. (Actually, the instructions for Invitrogen's Novex TBE-urea ones confuse me: one is usually advised to avoid storing urea gels at 4ºC due to precipitation, so I am susprised they say their gels can last weeks at that temperature. The longest I've ever let a urea-TBE gel sit is overnight at room temperature, for polymerization.)
Regardless of whether you buy precast gels or make them yourself, though, I can't imagine why you would want 2.5% acrylamide (?!) for 20-40nt miRNA, unless there's some technique downstream that explicitly requires ridiculously low acrylamide amounts. I would think that 6% (a pretty frequent polyacrylamide concentration for nucleic acids) would really be the absolute minimum, and you'd really want to go rather higher if your miRNA is in the 20-40nt range (which is only oligo size!) Probably closer to 15%, really. Just as higher percentages of agarose are used for resolving small things, higher percentages of acrylamide are used for resolving small things. Most manufacturers of electrophoresis stuff have guides that can help you choose the right percentage and so on, and which often include information on less-used techniques, the science behind electrophoresis, and so on. This is one I keep a pdf of due to the sheer number of techniques discussed, but National Diagnostics has a bunch of DNA/RNA-focused electrophoresis info on their site, there's a good chapter in a molecular bio book, etc.
But in the name of all that is holy ditch that evil EtBr!! Shiz gon' kill ya.
Meh. The problem with EtBr is mutagenicity, not toxicity. A lot of the newer alternatives are possibly less mutagenic (when there is data available), but have higher acute toxicity - and are often dissolved in DMSO, potentially making it easier for them to get into cells. (A post by a molecular biologist discussing this.) Anything that binds DNA has at least some potential to be problematic. Furthermore, any number of other things discussed here - formaldehyde, acrylamide, arguably the UV used to view EtBr gels - are more dangerous - but none of them should be a problem as long as you work safely, with proper protective equipment. Ethidium bromide is cheap and effective, and though there may be better options if you can afford them (for sensitivity and avoiding nucleic acid UV exposure, not just safety), it is highly unlikely to kill you.
posted by ubersturm at 1:49 PM on May 31, 2013 [1 favorite]
Response by poster: Yup, that EtBr is a mutagen, and so are so many things that we work with! My PI likes everything old school…we are very careful using it (nitrile gloves and change them every time we touch anything containing it) but I will definitely bring up the GelRed in our next meeting and see what he thinks.
I did try the Biotech. Molec. Bio forum a long while ago, and remember that it took about a week for my question to be answered, meanwhile, I just figured it out myself. :)
But I was browsing through it via your link, and yes, there is a lot of great information there.
And thanks also ubersturm for the EtBr article, it was very illuminating. That's a bookmarker, that is.
Thank you both very much!
posted by Arachnophile at 2:24 PM on May 31, 2013
I did try the Biotech. Molec. Bio forum a long while ago, and remember that it took about a week for my question to be answered, meanwhile, I just figured it out myself. :)
But I was browsing through it via your link, and yes, there is a lot of great information there.
And thanks also ubersturm for the EtBr article, it was very illuminating. That's a bookmarker, that is.
Thank you both very much!
posted by Arachnophile at 2:24 PM on May 31, 2013
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You can run 2-4% agarose gels no worries, USUALLY. They require more heat and time to get the agarose to all dissolve, and would note they set at a higher temperature so you have to pour them more quickly once molten.
These links may help:
http://link.springer.com/protocol/10.1385%2F1-59259-582-0%3A35
http://www.protocol-online.org/biology-forums/posts/19427.html
http://www.protocol-online.org/biology-forums-2/posts/12441.html
http://www.bio.net/bionet/mm/methods/1999-August/077812.html
You will be tempted to run them at a higher voltage to get them to give you a quick result, make sure you dont go too high lest your ohmic heating screw you.
Experimenting is your answer: likely the only way you will find out for sure is to do a test run with a suitably sized marker/ladder in one lane to make sure your gel is running OK, then use your precious miRNA in a diff gel.
posted by lalochezia at 3:02 PM on May 30, 2013 [1 favorite]