Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR
Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR
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DOI：
10.1242/jeb.01625
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发表时间：
2005-06-01
影响因子：
2.8
通讯作者：
Ito, E
中科院分区：
文献类型：
--
作者：
Wagatsuma, A;Sadamoto, H;Ito, E
基因表达在每个细胞中受到不同的调节，即使这些细胞包含在同一组织中。因此，我们需要测量单个细胞中mrna的数量，以便更好地了解转录机制。然而，目前还没有准确、快速和适当的方法来确定单个细胞中特定mrna的确切拷贝数。因此，我们开发了一种程序，用于分离单个可识别的细胞并确定其中mrna的确切拷贝数。我们首先分离了池塘蜗牛的大脑巨细胞，因为这个神经元在进食行为的联想学习所带来的学习行为的记忆巩固过程中起着关键作用。然后，我们确定了环状amp响应元件结合蛋白（creb）的mrna拷贝数。这些转录因子在动物物种的记忆形成中起着重要作用。该方案使用两种相互协调的技术：一种是用新开发的微操作器分离单个神经元的技术，另一种是通过定量实时逆转录聚合酶链反应（qRT-PCR）检测mrna。分子分析确定mRNA拷贝数，每个拷贝数与序列稀释的lynaea CREB mRNA对应的cDNA溶液制备的标准曲线进行比较。标准曲线在10 ~ 10(5)份范围内呈线性，测定内变异在15%以内。从神经节中取出的每个神经元直接穿刺提取总RNA，无需进一步纯化即可用于检测。通过这两步程序，我们发现CREB抑制因子（CREB2）的mRNA拷贝数在单个脑巨细胞中为30-240，而CREB激活因子（CREB1）的mRNA拷贝数低于检测限（< 25）。这些结果表明CREB级联是由脑巨细胞中过量的CREB2调节的。我们的程序是唯一的定量分析，以阐明基因转录的动态在一个单细胞。
Gene expression is differently regulated in every cell even though the cells are included in the same tissue. For this reason, we need to measure the amount of mRNAs in a single cell to understand transcription mechanism better. However, there are no accurate, rapid and appropriate methods to determine the exact copy numbers of particular mRNAs in a single cell. We therefore developed a procedure for isolating a single, identifiable cell and determining the exact copy numbers of mRNAs within it. We first isolated the cerebral giant cell of the pond snail Lymnaea stagnalis as this neuron plays a key role in the process of memory consolidation of a learned behavior brought about by associative learning of feeding behavior. We then determined the copy numbers of mRNAs for the cyclic AMP-responsive element binding proteins (CREBs). These transcription factors play an important role in memory formation across animal species. The protocol uses two techniques in concert with each other: a technique for isolating a single neuron with newly developed micromanipulators coupled to an assay of mRNAs by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The molecular assay determined the mRNA copy numbers, each of which was compared with a standard curve prepared from cDNA solutions corresponding to the serially diluted solutions of Lymnaea CREB mRNA. The standard curves were linear within a range of 10 to 10(5) copies, and the intra-assay variation was within 15%. Each neuron removed from the ganglia was punctured to extract the total RNA directly and was used for the assay without further purification. Using this two-step procedure, we found that the mRNA copy number of CREB repressor (CREB2) was 30-240 in a single cerebral giant cell, whereas that of CREB activator (CREB1) was below the detection limits of the assay (< 25). These results suggest that the CREB cascade is regulated by an excess amount of CREB2 in the cerebral giant cells. Our procedure is the only quantitative analysis for elucidation of the dynamics of gene transcription in a single cell.