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。% Q* J& |8 q$ _
  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~

nculsa747

茎环用固定的，只要加上设计的特异臂就行了。

86964109

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; _- A) p1 D) y& ?% q3 i 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 example$ _" Q" a3 e2 Q: V9 m8 ~- R

. C% q2 b% `3 ^( q! f5 TStem-loop RT primer for miR10a
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% T) B0 H+ Y. P+ V4 ^GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACcacaaa) g7 g9 Z) n! T
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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.4 _% v" y, j" R2 P
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>hsa-miR-10a 5'- UACCCUGUAGAUCCGAAUUUGUG -3'2 @$ p, x1 E+ b" V9 x& N/ T
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