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How to design probes for microRNA Northern Blot


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4 replies to this topic

#1 whitedolphin

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Posted 15 March 2009 - 04:57 AM

Any1 who experienced Northern Blot, is it ok to syntheze antisense of mature microRNA? OR I should carefully design it and analyze it using special software? Any suggestion and information is appreciated in advance! :D

#2 pcrman

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Posted 17 March 2009 - 09:50 PM

I guess the probe should be complementary to the mature miRNA because mature miRNA is what you are interested. You need to be cautious if the miRNA of your interest has "isoforms", ie, miRNAs with only a few nucleotide difference.

#3 whitedolphin

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Posted 20 March 2009 - 09:17 PM

I guess the probe should be complementary to the mature miRNA because mature miRNA is what you are interested. You need to be cautious if the miRNA of your interest has "isoforms", ie, miRNAs with only a few nucleotide difference.

Thanks! PCRman!
I found someone have used Probes not exactly comlementary to the mature miRNA,.i.e. , probes with 1 or 2 nt overhang when they hybrid to mature miRNA. I guess it may be useful when detect the mature miRNA but pre-miRNA/pri-miRNA. :D

#4 raja

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Posted 01 April 2009 - 10:30 PM

I was using antisense miR as a probe to hybridize, I can fish out pri, pre, and nature miRNA by Northern blot. I just had two "TT" at the end to avaid degradation.

it worked well for me.

#5 Fizban

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Posted 02 April 2009 - 02:02 AM

I was using antisense miR as a probe to hybridize, I can fish out pri, pre, and nature miRNA by Northern blot. I just had two "TT" at the end to avaid degradation.

it worked well for me.


What do u mean when you say "to avoid degradation"? why shoud your probe be degraded when u hybridize it on your filter? and how are 2 TT supposed to protect your probe?
BTW, do you use LNA or DNA probes? I've been using commercially available probes from exiqon but they're very expensive!
bye
Fizban




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