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miRNA knockdown approaches


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#1 Jimmyzxcd

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Posted 27 April 2011 - 03:45 PM

I would like to know if there is any cheap way to approach miRNA knockdown. These are the methods I have gathered so far:

1. Antisense oligonucleotides: There are lot of commercial options so far. Most of them are using modified nucleotides and charged for around $250/ea. I wonder if anyone tried using non-modified oligonucleotide for knockdown. The binding affinity may be lower but it's much cheaper and just like synthesizing primers with additional HPLC purification. Will it work?

2. siRNA against pri-miR: The idea is just like conventional siRNA mediated knockdown by degrading target mRNA, but, in this case, the target is pri-miR itself or the loop structure of pre-miR. I couldn't find out too much information about this approach, but this seems to be an economic alternative.

3. miRNA sponge: Construct a plasmid expressing the miRNA target 3'UTR sequence as a decoy to dilute the miR in cells. How long should a 3'UTR be included to be capable to work as an efficient decoy? Or I can just clone an antisense of mature-miRNA?

Any methods or ideas is appreciated. Thank you.

Jimmy

#2 Jon Moulton

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Posted 28 April 2011 - 07:58 AM

Citations for Morpholino oligos targeting miRNA:
http://www.gene-tool...#miRNAcitations
Jon D. Moulton
Gene Tools, LLC
www.gene-tools.com

#3 Jimmyzxcd

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Posted 28 April 2011 - 10:07 AM

Citations for Morpholino oligos targeting miRNA:
http://www.gene-tool...#miRNAcitations


I did check morpholinos, but it charges $400 per custom design. So far, minimum 300nmol is too much for me. What if the designed oligo doesn't work? I hope there is an option for smaller quantities, like 50nmol.

Jimmy

#4 Jon Moulton

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Posted 29 April 2011 - 07:57 AM


Citations for Morpholino oligos targeting miRNA:
http://www.gene-tool...#miRNAcitations


I did check morpholinos, but it charges $400 per custom design. So far, minimum 300nmol is too much for me. What if the designed oligo doesn't work? I hope there is an option for smaller quantities, like 50nmol.

Jimmy


Agreed, the minimum custom synthesis quantity is 300 nanomole. The oligos are not guaranteed to have the expected biological effect. Their success rate is good, but they do carry a risk.
Jon D. Moulton
Gene Tools, LLC
www.gene-tools.com

#5 pcrman

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Posted 29 April 2011 - 11:27 AM

Hi Jimmy,

If you want to find a cheap way of knocking down miRNA, probably targeting the loop sequence using standard siRNA is the way to go. You may need higher transfection concentration of the anti-miRNA siRNA because it functions in the nucleus and sometimes may need more than one siRNA targeting different location of the loop sequence.

Good luck.

#6 Earnest

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Posted 03 May 2011 - 05:23 PM

I would like to know if there is any cheap way to approach miRNA knockdown. These are the methods I have gathered so far:

1. Antisense oligonucleotides: There are lot of commercial options so far. Most of them are using modified nucleotides and charged for around $250/ea. I wonder if anyone tried using non-modified oligonucleotide for knockdown. The binding affinity may be lower but it's much cheaper and just like synthesizing primers with additional HPLC purification. Will it work?

2. siRNA against pri-miR: The idea is just like conventional siRNA mediated knockdown by degrading target mRNA, but, in this case, the target is pri-miR itself or the loop structure of pre-miR. I couldn't find out too much information about this approach, but this seems to be an economic alternative.

3. miRNA sponge: Construct a plasmid expressing the miRNA target 3'UTR sequence as a decoy to dilute the miR in cells. How long should a 3'UTR be included to be capable to work as an efficient decoy? Or I can just clone an antisense of mature-miRNA?

Any methods or ideas is appreciated. Thank you.

Jimmy


I'd go for transient or permanent infection using adenovirus or lentivirus constructs. They're more expensive, but the mirna targets have been validated and guaranteed to work.

http://www.abmgood.c...Adenovirus.html
http://www.abmgood.c...Lentivirus.html

If you have one known target, this would be the best way to go.

If you have multiple targets, siRNA constructs would work better and are definitely cheaper for screening:

http://www.abmgood.c...n=3710&dsn=3805

But once I found the one we wanted to study, we went with the lentivirus to do permanent infections. The miRNA knockdown did not result in a fatal phenotype.

#7 Earnest

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Posted 04 May 2011 - 06:26 AM


I would like to know if there is any cheap way to approach miRNA knockdown. These are the methods I have gathered so far:

1. Antisense oligonucleotides: There are lot of commercial options so far. Most of them are using modified nucleotides and charged for around $250/ea. I wonder if anyone tried using non-modified oligonucleotide for knockdown. The binding affinity may be lower but it's much cheaper and just like synthesizing primers with additional HPLC purification. Will it work?

2. siRNA against pri-miR: The idea is just like conventional siRNA mediated knockdown by degrading target mRNA, but, in this case, the target is pri-miR itself or the loop structure of pre-miR. I couldn't find out too much information about this approach, but this seems to be an economic alternative.

3. miRNA sponge: Construct a plasmid expressing the miRNA target 3'UTR sequence as a decoy to dilute the miR in cells. How long should a 3'UTR be included to be capable to work as an efficient decoy? Or I can just clone an antisense of mature-miRNA?

Any methods or ideas is appreciated. Thank you.

Jimmy


I'd go for transient or permanent infection using adenovirus or lentivirus constructs. They're more expensive, but the mirna targets have been validated and guaranteed to work.

http://www.abmgood.c...Adenovirus.html
http://www.abmgood.c...Lentivirus.html

If you have one known target, this would be the best way to go.

If you have multiple targets, siRNA constructs would work better and are definitely cheaper for screening:

http://www.abmgood.c...n=3710&dsn=3805

But once I found the one we wanted to study, we went with the lentivirus to do permanent infections. The miRNA knockdown did not result in a fatal phenotype.


I just got a promo email. P0009 gives 40% off every miRNA product. Might be worth a try.




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