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Old 03-10-2010, 01:46 PM   #1
kbushley
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Default mRNA fragmentation

Does anyone have an opinion whether the Ambion RNA fragmentation kit works well for RNA-SEQ. The documentation says it produces fragments of 60-200 bp. Any suggestions on parameters/incubation time to increase fragment size or other recommendation for chemical shearing?
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Old 03-11-2010, 06:34 AM   #2
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The ambion fragmentation kit will work fine. It is essentially a buffer containing Zn2+ as the metal ion, among other minor components. The ends of your fragmented RNA will not be compatible for ligation-based library prep methods until you further treat the fragmented RNA with T4 polynucleotide kinase and ATP. Chemical fragmentation results in 5' OH and 3' PO4; these ends need to be 'repaired' before ligation.

Fragmentation is inherently touchy. Different sources of RNA can demonstrate radically distinct time and temp requirements. I would recommend performing some pilot tests on your sample types using some basic parameters; vary temp between ~70-95oC, time between ~2-30 min. Use at least ~50 ng of RNA so you can easily see the fragmentation pattern on a bioanalyzer trace.

One thing to keep in mind about fragmented RNA. There is usually a fairly high molar quantity of 'short' fragments (<50 nt) that can dominate some library prep methods. These small products do not easily show up on gels or bioanalyzer trace since they tend not to stain well with SYBR Gold or Ethidium bromide. If you can afford to lose some material, it is worth doing some sort of RNA clean up after fragmentation. AMPure or SPRI beads are said to work fairly well, but yield can be somewhat low. Typically glass fiber filter methods (ie. Qiagen minelute) sometimes work too well and do not remove enough of the small material.

I hope this helps.
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Old 03-11-2010, 08:05 AM   #3
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Hi Scooter,

Thanks, that does help! Just wanted to make sure I'll be able to get a good size distribution with a little optimization.
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Old 08-23-2010, 11:51 PM   #4
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Quote:
Originally Posted by scooter View Post
The ambion fragmentation kit will work fine. It is essentially a buffer containing Zn2+ as the metal ion, among other minor components. The ends of your fragmented RNA will not be compatible for ligation-based library prep methods until you further treat the fragmented RNA with T4 polynucleotide kinase and ATP. Chemical fragmentation results in 5' OH and 3' PO4; these ends need to be 'repaired' before ligation.

Fragmentation is inherently touchy. Different sources of RNA can demonstrate radically distinct time and temp requirements. I would recommend performing some pilot tests on your sample types using some basic parameters; vary temp between ~70-95oC, time between ~2-30 min. Use at least ~50 ng of RNA so you can easily see the fragmentation pattern on a bioanalyzer trace.

One thing to keep in mind about fragmented RNA. There is usually a fairly high molar quantity of 'short' fragments (<50 nt) that can dominate some library prep methods. These small products do not easily show up on gels or bioanalyzer trace since they tend not to stain well with SYBR Gold or Ethidium bromide. If you can afford to lose some material, it is worth doing some sort of RNA clean up after fragmentation. AMPure or SPRI beads are said to work fairly well, but yield can be somewhat low. Typically glass fiber filter methods (ie. Qiagen minelute) sometimes work too well and do not remove enough of the small material.

I hope this helps.
Hi scooter,
I saw you are suggesting T4 PNK for RNA repair (5´kinase and 3´phosphatase)after fragmentation.Do you have any idea if the protocol that companies (we have T4PNK from Fermentas) are suggesting for 5´ phosphorilation works well also for 3´phosphatase?
thanks
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Old 08-24-2010, 11:13 AM   #5
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I think T4 PNK is a much better 5' kinase than 3' phosphatase, but clearly has both activities. After chemical fragmentation the 3' ends are a mixture of 3' PO4, 2' PO4 or 2'-3' cyclic phosphate at some unknown ratio of the three. This would mean that some 3' ends are already compatible for ligation (the ends with 2' PO4 will have a 3' OH) and PNK will then repair the ends with a 3' PO4 (but not the cyclic ends). Thus, even with a somewhat weak phosphatase activity you will still be able to recover a decent proportion of your 3' ends.

I think most company protocols should work fine. However, I think the phosphatase activity is likely a bit better in the absence of ATP, but of course the kinase activity won't function under these conditions.

I hope that is helpful.
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Old 08-25-2010, 07:47 AM   #6
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thank you scooter!very helpful, I have to consider this in setting the T4 PNK reaction!
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Old 08-16-2011, 10:28 AM   #7
Argonaute
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Any recommendations on conditions to maximize 5' kinase and 3' phosphatase activity? I was wondering if anyone has found a highly tuned protocol?
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Old 10-18-2011, 09:26 AM   #8
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Quote:
Originally Posted by scooter View Post
I think T4 PNK is a much better 5' kinase than 3' phosphatase, but clearly has both activities. After chemical fragmentation the 3' ends are a mixture of 3' PO4, 2' PO4 or 2'-3' cyclic phosphate at some unknown ratio of the three. This would mean that some 3' ends are already compatible for ligation (the ends with 2' PO4 will have a 3' OH) and PNK will then repair the ends with a 3' PO4 (but not the cyclic ends). Thus, even with a somewhat weak phosphatase activity you will still be able to recover a decent proportion of your 3' ends.

I think most company protocols should work fine. However, I think the phosphatase activity is likely a bit better in the absence of ATP, but of course the kinase activity won't function under these conditions.

I hope that is helpful.
Your post is so informative, thanks for sharing!
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Old 10-27-2011, 08:02 AM   #9
perrinwang
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We use first-strand buffer from SuperScript II/III to perform the fragmention as this eliminate the need of purification before reverse-transcription.

http://dx.plos.org/10.1371/journal.pone.0026426

Our paper described what the profile of sheared RNA molecules... the smaller fragments (e.g <100) can be effectively removed using SPRI purification.
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Old 01-23-2012, 08:15 AM   #10
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Hello all,

Can anyone tell me why in some protocols (Directional mRNA-seq) one has to treat with a phosphatase (that removes de 5' phosphate) and then with T4 PNK after RNA fragmentation? I mean, it seems to me that treatment with T4 PNK would be enough to repair the 5'OH and the 3'P ends, so what is the point of treating with a phosphatase?

Sorry if the question is too obvious.

Thanks in advance!
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Old 01-24-2012, 04:02 AM   #11
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Quote:
Originally Posted by amazonic9 View Post
Hello all,

Can anyone tell me why in some protocols (Directional mRNA-seq) one has to treat with a phosphatase (that removes de 5' phosphate) and then with T4 PNK after RNA fragmentation? I mean, it seems to me that treatment with T4 PNK would be enough to repair the 5'OH and the 3'P ends, so what is the point of treating with a phosphatase?

Sorry if the question is too obvious.

Thanks in advance!
I think some protocols ligate a single-stranded 3' adapter to the RNA first using T4 RNA ligase 2, then T4 PNK and then ligate the 5' adapter? If so, then the reason is that they don't want the 3' adapter ligating to the 5' end of the RNA. So they dephosphorylate it. They could make the 3' adapter 3'-dideoxy instead, I guess. But maybe they do both.

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Old 01-25-2012, 07:55 AM   #12
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Thank you phillip for the answer. That makes sense to me. But in the particular case that I am using this phosphatase + PNK treatment, do not ligate a 3' adapter before the treatment with PNK. The workflow is: fragmentation of RNA, treatment with phosphatase, treatment with PNK, and then ligation of the 3' and 5' adapters. So I still do not understand what is the sense of using that phosphatase step...

Any other idea?

Thank you!

amazonic9
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Old 01-25-2012, 08:51 AM   #13
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The lore is that T4 PNK's 3' phosphatase activity is "weak". I think heat + divalent cation based fragmentation methods yield 5' hydroxyl/3' phosphate and this must be reversed for T4 ligases to work. So it seems likely to me that your protocol decided to err on the side of caution in removing the 3' phosphates.

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Old 01-25-2012, 11:10 AM   #14
Garyron
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I had the same question as amazonic9 since I also came across the same protocol. Thanks so much Phillip for your helpful post.
Antartic phosphatase will remove the 5'P along with 3'P, Right? and then PNK will add 5'P and also remove 3'P. Instead if we just carry out PNK treatment ( more units than usual) i in absence of ATP, will it not have the same effect?
Thanks
J
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Old 01-25-2012, 12:22 PM   #15
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In principle, yes. But I have never attempted it myself, so be forewarned. Lots of things that work in principle fail to work in practice. But if it is bugging you, it would be worth an attempt.

Alternatively you dig up a paper on T4 PNK activities. Sometimes the issue is that one activity works under one set of conditions while another is optimum under a different set.

Oh, one more issue. From what I read in the past the major product of heat/divalent cation RNA fragmentation are fragments with a 3'-2' cyclic phosphate group. Could be this hydrolyzes at a high rate into either a plain 3' phosphate or a 2' phosphate. Or not. Can T4PNK remove a cyclic phosphate? Can Shrimp Alkaline Phosphatase remove a cyclic phosphate? I don't know.

The problem with altering a protocol that is working is that you venture off the "path" that someone else has blazed for you. It might be a poor path, but if you have gotten it to work in the past, you have evidence that it will get you where you need to go. Venture off the path and you are more or less on your own...

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Old 01-28-2012, 05:37 AM   #16
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T4 PNK 3'dephosphorylaion protocol has been used by many which dephosphorylates all Phosphate groups ( not 5'P), 12.5 units T4 PNK (New England Biolabs) and MES-NaOH buffer (100 mM MESNaOH pH 5.5, 10 mM MgCl2, 10 mM β-mercaptoethanol, 300 mM NaCl) for 6 h at 37°C
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Old 01-30-2012, 06:29 AM   #17
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Again, thank you for your answers. I think this sentence could be the key "Sometimes the issue is that one activity works under one set of conditions while another is optimum under a different set."
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