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Problems with amplifying microRNA


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#1 angelapark

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Posted 24 January 2011 - 12:03 PM

Whenever I do PCR (not real time) on miRNA, only half of my samples get amplified while the other half don't work.

For example, today I ran gel electrophoresis on mmu-mir-466d, mmu-mir-1-2-as, mmu-mir-466a, and mmu-mir 466c
but only saw intense and correct band (~500bp)for mir-1-2-as and mir-466c. For the other two, I saw faint single band that was around ~300bp.

Is anyone having similar problems in amplifying miRNA? What can I do to fix this problem?

Thank you very much!

#2 UBClabbie

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Posted 24 January 2011 - 02:29 PM

Hmm... maybe u need to optimize your PCR? Can you give more background on reagents, duration and setup?
Are you probing for pri-microRNA? bc 500bp and 300 bp isn't mature microRNA size. i can reccommend primers for mature miRNA, send me a message if u need more deets.

#3 angelapark

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Posted 24 January 2011 - 03:09 PM

Thank you so much for your reply!

I used mouse genomic DNA to amplify (DNA that code for those miRNA)and using that PCR product as an insert for subcloning.

For reagents, I had water, template, 2*Taq plus, and primer mix.
Then used "0.5KB" program on PCR machine. the program runs for about 1hr and 40 minutes.
The annealing temp is 50C and denaturing temp is 94C.




Hmm... maybe u need to optimize your PCR? Can you give more background on reagents, duration and setup?
Are you probing for pri-microRNA? bc 500bp and 300 bp isn't mature microRNA size. i can reccommend primers for mature miRNA, send me a message if u need more deets.



#4 UBClabbie

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Posted 26 January 2011 - 04:50 PM

Thank you so much for your reply!

I used mouse genomic DNA to amplify (DNA that code for those miRNA)and using that PCR product as an insert for subcloning.

For reagents, I had water, template, 2*Taq plus, and primer mix.
Then used "0.5KB" program on PCR machine. the program runs for about 1hr and 40 minutes.
The annealing temp is 50C and denaturing temp is 94C.





Hmm... maybe u need to optimize your PCR? Can you give more background on reagents, duration and setup?
Are you probing for pri-microRNA? bc 500bp and 300 bp isn't mature microRNA size. i can reccommend primers for mature miRNA, send me a message if u need more deets.



Hmm your annealing temperature seems really low to me for a product that long.... Maybe you are having a specificity issue? Try a 3 step program maybe and raise the annealing temperature? What are the length of your primers and what are the Tm of your primers?




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