Cell Sorting Physical Measurement

现有的mRNA定量方法 随着越来越明显的是，个体细胞中的基因表达明显偏离细胞群体的平均行为(Raj和van Oudenaarden，2008)，需要新的方法来提供个体细胞中mRNA拷贝数的准确整数计数。理想情况下，这种方法还应该揭示mRNAs在细胞内的位置，因为mRNAs的定位通常被细胞用来在空间上限制 RNA结合蛋白的活性(St Johnston，2005)。布鲁姆实验室(比奇等人，1999)和Robert Singer及其同事(Bertrand等人，1998)同时开发的MS2信使核糖核酸检测技术是足以灵敏地检测单个mRNA分子的方法之一。在这种方法中，基因被设计成转录一种在其未翻译区域包含特定RNA发夹的许多拷贝的mRNA， 每一个都与噬菌体MS2的外壳蛋白紧密结合。然后，这个基因在一个已经表达了融合了GFP的MS2外壳蛋白的细胞中表达。当许多MS2-GFP蛋白与单个mrna结合时，会产生足够的荧光信号，使得使用传统的荧光显微镜可以检测到单个mrna作为衍射限制的斑点，从而使人们能够计数 单个细胞中的mRNA。尽管这项技术提供了关于mRNAs的定量和空间信息，但它的使用受到两个实质性限制的阻碍：第一，目标生物体需要进行基因改造；第二，不可能检测到超过一个mRNA物种。此外，增加串联的MS2结合位点显著改变了RNA的稳定性，MS2-GFP融合蛋白往往聚集在大簇中，阻碍了单分子的检测。

Existing mRNA quantification methods As it is becoming increasingly apparent that gene expression in individuai cells deviates substantially from the average behavior of cell populations (Raj and van Oudenaarden, 2008), new methods that provide accurate integer counts of mRNA copy numbers in individual cells are needed. Ideally, such methods should also reveal the intracellular locations of the mRNAs, as mRNA localization is often used by cells to spatially restrict the activity of RNA binding proteins (St Johnston, 2005). One of the methods sensitive enough to detect single mRNA molecules is the MS2 mRNA detection technique developed simultaneously by the Bloom laboratory (Beach et al., 1999) and Robert Singer and colleagues (Bertrand et al., 1998). In this method, a gene is engineered to transcribe an mRNA containing many copies of a specific RNA hairpin in its untranslated region, each of which binds tightly to the coat protein of the bacteriophage MS2. This gene is then expressed in a cell that already expresses the MS2 coat protein fused to GFP. When many of the MS2-GFP proteins bind to an individual mRNA, enough fluorescent signal is generated that the individual mRNAs are detectable as diffraction limited spots using conventional fluorescence microscopy, allowing one to count the number of mRNAs in single cells. Although this technique provides quantitative and spatial information about mRNAs, its use is hindered by two substantial limitations: first, the target organism needs to be genetically modified; and second, detection of more than one mRNA species is impossible. Additionally, adding the tandem MS2 binding sites signiflcantly changes the RNA stability and the MS2-GFP fusion proteins tends to aggregate in large clusters hindering the detection of single molecules.