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pre-miRNA existence


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#1 j123

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Posted 26 January 2010 - 03:31 PM

I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.

#2 Fizban

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Posted 27 January 2010 - 01:22 AM

I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.


You have 2 ways:
1) you can use in situ hybridization with probes specific for both pre and pri miRNA. LNA probes probably better. i saw some very interesting results in cambridge 2 years ago, you could have surprising results, keep us up to date!

2) qRT-PCR. This is a littlebit tricky. Precursors have hairpin structures so they are not good candidates for primer design. u have to cope with it. i managed to design working primers for my miRNA of interest, it took some work and stratagene Herculase to work with complex regions, but its feasible.
if i remember well it's possible that Qiagen sells PRE miRNA ready primer sets but u have to check.

probably others have better ideas but that's all i can think now.
i'd not try northern blot, it could be a waste of time if your miRNA is not strongly expressed but this is a personal opinion.
good luck
Fiz

#3 j123

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Posted 27 January 2010 - 07:12 AM

Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.


You have 2 ways:
1) you can use in situ hybridization with probes specific for both pre and pri miRNA. LNA probes probably better. i saw some very interesting results in cambridge 2 years ago, you could have surprising results, keep us up to date!

2) qRT-PCR. This is a littlebit tricky. Precursors have hairpin structures so they are not good candidates for primer design. u have to cope with it. i managed to design working primers for my miRNA of interest, it took some work and stratagene Herculase to work with complex regions, but its feasible.
if i remember well it's possible that Qiagen sells PRE miRNA ready primer sets but u have to check.

probably others have better ideas but that's all i can think now.
i'd not try northern blot, it could be a waste of time if your miRNA is not strongly expressed but this is a personal opinion.
good luck
Fiz



#4 Fizban

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Posted 27 January 2010 - 08:47 AM

Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


Here in italy custom probes design and validation is available. you have just to wait for validation of your probes but shouldn't take long.
If it works in italy....it works everywhere!
:D sad but almost always true
bye Fiz

#5 j123

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Posted 27 January 2010 - 10:47 AM

Hi, Fizban, could you please send me the link? Thank you very much for the helpful discussion.


Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


Here in italy custom probes design and validation is available. you have just to wait for validation of your probes but shouldn't take long.
If it works in italy....it works everywhere!
:D sad but almost always true
bye Fiz



#6 Fizban

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Posted 27 January 2010 - 11:52 PM

news from ABI: they have PRI-miRNA probes!
https://products.app...mp;catID=607040

Actually there's no link for mirna custom assays on the website, just contact your local ABI representative and ask. they have stock of inventoried assays and made-to-order assays. you give them the sequence and they'll give you a VALIDSTED mirna assay. that's how it worked for us with probes made for zebrafish.

hope it helps
Fiz

#7 cathywill

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Posted 28 January 2010 - 11:36 AM

Yes, they can make customerized Taqman probe for you. It costs about $230, and a week's wait. Use their website for ordering.

Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.


You have 2 ways:
1) you can use in situ hybridization with probes specific for both pre and pri miRNA. LNA probes probably better. i saw some very interesting results in cambridge 2 years ago, you could have surprising results, keep us up to date!

2) qRT-PCR. This is a littlebit tricky. Precursors have hairpin structures so they are not good candidates for primer design. u have to cope with it. i managed to design working primers for my miRNA of interest, it took some work and stratagene Herculase to work with complex regions, but its feasible.
if i remember well it's possible that Qiagen sells PRE miRNA ready primer sets but u have to check.

probably others have better ideas but that's all i can think now.
i'd not try northern blot, it could be a waste of time if your miRNA is not strongly expressed but this is a personal opinion.
good luck
Fiz



#8 j123

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Posted 28 January 2010 - 12:55 PM

Thanks a lot for the information. I really appreciate it.

#9 j123

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Posted 28 January 2010 - 12:59 PM

I check AB website, there are two kinds of probes :
* TaqManŽ MGB Probes
* TaqManŽ TAMRA™ Probes

Could you please tell me which one I should order? Thanks again for your kind help.





Yes, they can make customerized Taqman probe for you. It costs about $230, and a week's wait. Use their website for ordering.

Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.


You have 2 ways:
1) you can use in situ hybridization with probes specific for both pre and pri miRNA. LNA probes probably better. i saw some very interesting results in cambridge 2 years ago, you could have surprising results, keep us up to date!

2) qRT-PCR. This is a littlebit tricky. Precursors have hairpin structures so they are not good candidates for primer design. u have to cope with it. i managed to design working primers for my miRNA of interest, it took some work and stratagene Herculase to work with complex regions, but its feasible.
if i remember well it's possible that Qiagen sells PRE miRNA ready primer sets but u have to check.

probably others have better ideas but that's all i can think now.
i'd not try northern blot, it could be a waste of time if your miRNA is not strongly expressed but this is a personal opinion.
good luck
Fiz



#10 cathywill

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Posted 28 January 2010 - 01:20 PM

I ordered the MGB probe with FAM Dye, since it binds to the loop part of the pre-miRNA. It is a hydrolysis probe which will give out signal at the elongation stage of the PCR.
Of course you will need sense and antisense primers binding to the 5' and 3' end of the pre-miRNA sequence, respectively. Check the guideline for primer design. They have relative strict rules for the sequence and Tm for both primers and probe.
Not familiar with TAMRA probe.

I check AB website, there are two kinds of probes :
* TaqManŽ MGB Probes
* TaqManŽ TAMRA™ Probes

Could you please tell me which one I should order? Thanks again for your kind help.





Yes, they can make customerized Taqman probe for you. It costs about $230, and a week's wait. Use their website for ordering.

Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.


You have 2 ways:
1) you can use in situ hybridization with probes specific for both pre and pri miRNA. LNA probes probably better. i saw some very interesting results in cambridge 2 years ago, you could have surprising results, keep us up to date!

2) qRT-PCR. This is a littlebit tricky. Precursors have hairpin structures so they are not good candidates for primer design. u have to cope with it. i managed to design working primers for my miRNA of interest, it took some work and stratagene Herculase to work with complex regions, but its feasible.
if i remember well it's possible that Qiagen sells PRE miRNA ready primer sets but u have to check.

probably others have better ideas but that's all i can think now.
i'd not try northern blot, it could be a waste of time if your miRNA is not strongly expressed but this is a personal opinion.
good luck
Fiz


Edited by cathywill, 28 January 2010 - 01:21 PM.


#11 j123

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Posted 28 January 2010 - 01:49 PM

Thanks a lot. It really helps.


I ordered the MGB probe with FAM Dye, since it binds to the loop part of the pre-miRNA. It is a hydrolysis probe which will give out signal at the elongation stage of the PCR.
Of course you will need sense and antisense primers binding to the 5' and 3' end of the pre-miRNA sequence, respectively. Check the guideline for primer design. They have relative strict rules for the sequence and Tm for both primers and probe.
Not familiar with TAMRA probe.

I check AB website, there are two kinds of probes :
* TaqManŽ MGB Probes
* TaqManŽ TAMRA™ Probes

Could you please tell me which one I should order? Thanks again for your kind help.





Yes, they can make customerized Taqman probe for you. It costs about $230, and a week's wait. Use their website for ordering.

Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.


You have 2 ways:
1) you can use in situ hybridization with probes specific for both pre and pri miRNA. LNA probes probably better. i saw some very interesting results in cambridge 2 years ago, you could have surprising results, keep us up to date!

2) qRT-PCR. This is a littlebit tricky. Precursors have hairpin structures so they are not good candidates for primer design. u have to cope with it. i managed to design working primers for my miRNA of interest, it took some work and stratagene Herculase to work with complex regions, but its feasible.
if i remember well it's possible that Qiagen sells PRE miRNA ready primer sets but u have to check.

probably others have better ideas but that's all i can think now.
i'd not try northern blot, it could be a waste of time if your miRNA is not strongly expressed but this is a personal opinion.
good luck
Fiz



#12 lihkin

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Posted 21 February 2010 - 02:14 AM

Hi,
I too have got a miRNA candidate that I have validated by northern. But I get 2 close bands at the top(sharp and above U6 band) and Mostly 1 prominent but a thicker band above 25nt. I have a few questions here -
1. Are the top two band pre-miRNA bands. If so why two bands.
2.What is the size of U6 RNA(plants).
3.What marker apart from NEB miRNA marker I can use for size determination of all these bands in a 15% denaturing gel.
Thanks,
lihkin
(PS: I am just a new member and did not know how to post so wrote in here,when I saw something relevant to my question).
Thanks,
Lihkin

#13 Noumy

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Posted 01 June 2010 - 05:30 AM

Hi!

I also would like to detect a precursor, but for artificial miRNAs. How can I validate the expression of an amiRNA? Do I also have to show the expression of its precursor? If yes, is an RT-PCR sufficient for the precursor?

Thanks in advance for your help!

#14 Fizban

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Posted 07 June 2010 - 03:04 AM

Hi!

I also would like to detect a precursor, but for artificial miRNAs. How can I validate the expression of an amiRNA? Do I also have to show the expression of its precursor? If yes, is an RT-PCR sufficient for the precursor?

Thanks in advance for your help!


well that should be easier than when coping with "natural" miRNAs. the good part is that you have the sequence and that it should differ from any other miRNA. bad part: the precursor is, for definition, an hairpin so not the best substrate for PCR primers, bau that can be done. many vendors now create the primers for you needing only the sequence and what u want to amplify, that could be a good choice. the other way around is to do northern blot, maybe with LNA probes, again, custom made.
you just have to check your wallet to decide which one is the best option! :P
Fiz




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