请教miRNA茎环引物设计的具体注意问题或设计方法

foodmic

    最近正在学习miRNA RT-PCR，对于其茎环引物和其上下游引物的设计是一个重要问题。
  J: u% k) \2 m2 @  他们的设计原理我是理解的，但是具体的设计问题很混乱。比如说，茎环引物的茎和环需要多长、Tm值等等；上游引物的尾巴多长、与miRNA重合碱基的个数；下游引物来自茎部和环的碱基个数多少，等等等等。请茎环引物专家多多赐教~

86964109

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1 _2 V6 H) U9 v. t Real-Time PCR Quantification of Plant miRNAs Using Universal ProbeLibrary Technology.pdf (219.14 KB)

2012-9-12 13:16 上传

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Here is an example1 s3 _. o) s# d6 `3 `2 f  h( E

% R: C2 x# w. j. `2 @. X! oStem-loop RT primer for miR10a& M6 u2 h8 i" `  ^* y

- X$ Q0 _! y# I8 c5 CGTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACcacaaa
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In the RT primer, only the last 6 nt are unique to miR-10a, and will bind to the 3' end (green) of miR-10a. The rest (44-nt) of it will form stem-loop structure. The purpose of stem-loop is to increase melting temperature. You can use the same sequence for many miRNAs, but the last 6 nt must be specific to the miRNA you want to detect.
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>hsa-miR-10a 5'- UACCCUGUAGAUCCGAAUUUGUG -3'
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nculsa747

茎环用固定的，只要加上设计的特异臂就行了。