Stem-loop RT-qPCR for miRNAs.
Stem-loop RT-qPCR for miRNAs.
复制标题
miRNA 的茎环 RT-qPCR。
DOI：
10.1002/0471142727.mb1510s95
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发表时间：
2011-07
影响因子：
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通讯作者：
Kramer, Martha F
中科院分区：
文献类型：
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作者：
Kramer, Martha F
本单元介绍了一种特异性和敏感性的定量逆转录PCR（RT-qPCR）方法，用于测量组织或培养细胞中的单个microRNA（miRNAs）。miRNA的长度为17 - 24个核苷酸（nt）。标准和定量PCR方法需要的模板长度至少是特异性正向或反向引物的两倍，每个引物的长度通常为20 nt。因此，靶最小长度为≥ 40 nt，使得miRNA对于标准RT-qPCR方法来说太短。在本试验中，每种RT-qPCR核酸试剂（包括RT引物、正向和反向PCR引物以及水解探针）均包含设计特征，这些设计特征共同优化了miRNA特异性和试验灵敏度。RT引物含有高度稳定的茎环结构，其延长靶cDNA。正向PCR引物增加了额外的长度，并具有优化其解链温度（Tm）并增强测定特异性的核苷酸。反向引物破坏茎环。通过将探针放置在大部分原始miRNA序列上来进一步优化测定特异性，并且通过添加小沟结合（MGB）部分来优化探针Tm。
This unit presents a specific and sensitive quantitative reverse-transcription PCR (RT-qPCR) method for measuring individual microRNAs (miRNAs) in tissue or cultured cells. MiRNAs are 17 – 24 nucleotides (nt) in length. Standard and quantitative PCR methods require a template that is at least twice the length of either of the specific forward or reverse primers, each typically ∼ 20 nt in length. Thus, the target minimum length is ≥ 40 nt, making miRNAs too short for standard RT-qPCR methods. In this assay, each of the RT-qPCR nucleic acid reagents, including the RT-primer, the forward and reverse PCR primers, and the hydrolysis probe, contain design features that, together, optimize miRNA specificity and assay sensitivity. The RT-primer contains a highly stable stem-loop structure that lengthens the target cDNA. The forward PCR primer adds additional length with nucleotides that optimize its melting temperature (Tm) and enhance assay specificity. The reverse primer disrupts the stem loop. Assay specificity is further optimized by placement of the probe over much of the original miRNA sequence, and the probe Tm is optimized by addition of a minor groove binding (MGB) moiety.