Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells.
Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells.
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DOI：
10.1093/nar/gkm593
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发表时间：
2007
影响因子：
14.9
通讯作者：
Tsourkas A
中科院分区：
文献类型：
--
作者：
Chen AK;Behlke MA;Tsourkas A
越来越多的研究使用分子信标（MBs）对活细胞中的RNA表达进行成像；然而，非特异性相互作用和/或核酸酶降解产生的假阳性信号会阻碍进行准确测量的能力。在本研究中，我们发现这种非特异性信号仅出现在活细胞的细胞核中。当通过将分子信标连接到量子点（QDs）使其保留在细胞质区室时，假阳性信号降低到边缘水平。因此，利用分子信标 - 量子点偶联物通过量化单个细胞发出的总荧光信号来测量MCF - 7乳腺癌细胞中内源性原癌基因c - myc的表达。添加他莫昔芬后，分子信标荧光的测量结果表明c - myc表达降低了71%，这与RT - PCR测量结果高度相关。通过每天对荧光校准标准品进行成像来解释仪器波动导致的分子信标荧光变化。此外，已确定总荧光信号的测量对焦平面不敏感。总体而言，这些结果提供了证据，表明当分子信标保留在细胞质中时，可以对RNA水平进行准确测量。
There have been a growing number of studies where molecular beacons (MBs) are used to image RNA expression in living cells; however, the ability to make accurate measurements can be hampered by the generation of false-positive signals resulting from non-specific interactions and/or nuclease degradation. In the present study, we found that such non-specific signals only arise in the nucleus of living cells. When MBs are retained in the cytoplasmic compartment, by linking them to quantum dots (QDs), false-positive signals are reduced to marginal levels. Consequently, MB–QD conjugates were used to measure the expression of the endogenous proto-oncogene c-myc in MCF-7 breast cancer cells by quantifying the total fluorescent signal emanating from individual cells. Upon the addition of tamoxifen, measurements of MB fluorescence indicated a 71% reduction in c-myc expression, which correlated well with RT-PCR measurements. Variations in MB fluorescence resulting from instrumental fluctuations were accounted for by imaging fluorescent calibration standards on a daily basis. Further, it was established that measurements of the total fluorescent signal were not sensitive to the focal plane. Overall, these results provide evidence that accurate measurements of RNA levels can be made when MBs are retained in the cytoplasm.
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DOI：
10.1073/pnas.2233244100
发表时间：
2003-11-11
影响因子：
11.1
作者：
Bratu, DP;Cha, BJ;Tyagi, S
通讯作者：
Tyagi, S
影响因子：
7.4
作者：
Medley, CD;Drake, TJ;Tan, WH
通讯作者：
Tan, WH
影响因子：
3.3
作者：
Cady, Nathaniel C.;Strickland, Aaron D.;Batt, Carl A.
通讯作者：
Batt, Carl A.
影响因子：
2.9
作者：
Henry, MR;Stevens, PW;Kelso, DM
通讯作者：
Kelso, DM
DOI：
10.1073/pnas.94.3.814
发表时间：
1997-02-04
影响因子：
11.1
作者：
McAdams, HH;Arkin, A
通讯作者：
Arkin, A