Multiplexed miRNA northern blots via hybridization chain reaction.
Multiplexed miRNA northern blots via hybridization chain reaction.
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DOI：
10.1093/nar/gkw503
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发表时间：
2016-09-06
影响因子：
14.9
通讯作者：
Pierce NA
中科院分区：
文献类型：
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作者：
Schwarzkopf M;Pierce NA
Northern blots可以使用标准台式设备检测生物样品中感兴趣的靶RNA。mirna是最具挑战性的靶标，因为它们必须用单个短核酸探针检测。在现有的方法中，同时检测多个rna的多重印迹很麻烦，阻碍了相互作用调控元件的研究。在这里，我们通过展示基于杂交链反应（HCR）机制的多路northern印迹来解决这一缺点。通过这种方法，与RNA靶点互补的核酸探针触发连锁反应，其中荧光团标记的DNA发夹自组装成拴在一起的荧光扩增聚合物。HCR的可编程性允许多个放大器在一个印迹内同时独立操作，从而实现直接的多路复用。我们展示了在293T和HeLa细胞提取的总RNA中同时检测到三种内源性miRNAs。对于给定的靶标，HCR信号随靶标丰度线性扩展，从而实现相对和绝对定量。使用非放射性HCR，使用2'OMe-RNA探针实现了敏感和选择性的miRNA检测。HCR northern blot方案需要~ 1.5天，与目标rna的数量无关。
Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2′OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs.
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DOI：
10.1073/pnas.0407024101
发表时间：
2004-10-26
影响因子：
11.1
作者：
Dirks, RM;Pierce, NA
通讯作者：
Pierce, NA
影响因子：
14.9
作者：
Válóczi, A;Hornyik, C;Havelda, Z
通讯作者：
Havelda, Z
影响因子：
3.3
作者：
Rarnkissoon, SH;Mainwaring, LA;Kajigaya, S
通讯作者：
Kajigaya, S
DOI：
10.1073/pnas.74.12.5350
发表时间：
1977-01-01
影响因子：
11.1
作者：
ALWINE, JC;KEMP, DJ;STARK, GR
通讯作者：
STARK, GR
影响因子：
46.9
作者：
通讯作者：
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