Cell Sorting Physical Measurement

现有mRNA定量方法 随着个体细胞中的基因表达显著偏离细胞群体的平均行为变得越来越明显（Raj和货车Oudenaarden，2008），需要提供个体细胞中mRNA拷贝数的精确整数计数的新方法。理想地，此类方法还应揭示mRNA的细胞内位置，因为mRNA定位通常被细胞用于在空间上限制mRNA的表达。 RNA结合蛋白的活性（St约翰斯顿，2005）。检测单个mRNA分子的足够灵敏的方法之一是由Bloom实验室同时开发的MS 2 mRNA检测技术（海滩et al.，1999）和Robert Singer及其同事（Bertrand等人，1998年）。在该方法中，基因被改造以转录在其非翻译区中含有多个拷贝的特定RNA发夹的mRNA， 每一个都与噬菌体MS 2的外壳蛋白紧密结合。然后，该基因在已经表达与GFP融合的MS 2外壳蛋白的细胞中表达。当许多MS 2-GFP蛋白与单个mRNA结合时，会产生足够的荧光信号，使得使用传统荧光显微镜可以将单个mRNA检测为衍射极限斑点，从而允许人们计数mRNA的数量。 单细胞中的mRNA。虽然这种技术提供了有关mRNA的定量和空间信息，但其使用受到两个实质性限制的阻碍：第一，靶生物需要进行遗传修饰;第二，不可能检测到一种以上的mRNA。此外，添加串联的MS 2结合位点显著改变RNA稳定性，并且MS 2-GFP融合蛋白倾向于聚集成大簇，阻碍单个分子的检测。

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.