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1、荧光条形码标记的单分子检测技术是一种全新的高通量检测基因表达谱、miRNA 等分子的技术，其定量结果可与 real-time PCR 相媲美。由于它的高通量和高精确性填补了基因芯片和 real-time PCR 之间的技术空白，一经问世大受欢迎，其检测结果频频登载在 nature、science 、cell 等顶级杂志上。NanoString 公司研发的荧光条形码标记的单分子检测技术最早诞生于 Leroy Hood 博士创立的系统生物学研究所（Institute for Systems Biology, ISB），该技术因可大批量检测多个样本多个基因，且定量精准而颇受瞩目，2008 年被Nat
2、ure Technology 杂志以封面文章的形式报道，报道如下：基因表达谱的检测目前有两种方法：基因芯片和 real-time PCR。但它们都需要逆转录或其它的酶促反应，这在一定程度上增加了人为操作及实验反应的误差。因此，Geiss 等人研发了全新的直接测定读取 mRNA 表达量的NanostringnCounter 分析系统，无须逆转录及其它酶促反应，只需 100ng 的总RNA 即可检测，具有极高的灵敏度和可重复性，它的出现成为科研工作者除基因芯片和 real-time PCR 之外的新选择。NanostringnCounter 仪器的实验原理是设计出捕捉探针和报告探针两种探针，分别与
3、靶 mRNA 结合。其中捕捉探针标有生物素，用于将杂交复合物固定在cartridge 板上；报告探针则带有四色荧光集团，其不同的排列组合方式代表不同的基因。将总 RNA 与这两种探针进行杂交、纯化、固定，扫描报告探针尾部的荧光集团确定其捕获的基因及其表达量。由于基因表达量的鉴定是由荧光集团的排列组合决定的，背景噪音不会影响基因的定量，因此该方法与基因芯片相比具有极高的灵敏度，可用来检测极低丰度的 mRNA，即使每个细胞只表达单个 mRNA 也能够被检测出来，且其精确性与 real-time PCR 相当，但通量则比 real-time PCR 高得多。在 NanostringnCounter
4、仪器中设计的探针长约 35-50 个碱基，且 mRNA 要与捕捉探针和报告探针两条探针同时结合才能被检测到，从而降低了实验的假阳性率。其中，标有生物素的捕捉探针会与 cartridge 板上的亲和链霉素结合，为杂交复合物的荧光扫描做准备；报告探针之后则有 6 个荧光探针位点，用于四色荧光标记，根据不同的排列组合，他们最多可标记 46，即 4096 个探针。排除容易混淆的荧光探针和需要做阳性对照和阴性对照的荧光探针，该仪器一次可标记 800 个荧光探针做目标 mRNA 的检测。NanostringnCounter 仪器已和 Affymetrix 芯片进行比较。经过检测一系列宽动态范围的 mRNA
5、 表达，结果显示两者之间变异系数 20%，并且有些低丰度mRNA 可在 nanostringnCounter 仪器中检出而在 Affymetrix 芯片中检测不出来。NanostringnCounter 仪器和基因表达串联分析（serial analysis of gene expression, SAGE）技术相似，但 nanostring 有更多优势，例如液相杂交，高通量检测，靶mRNA 无须扩增。与 real-time PCR 相比，nanostring 只需单步反应，比 real-time PCR 操作更简便，且通量也比 real-time PCR 更高。总之，NanostringnC
6、ounter 仪器所需样本更少、无需酶反应、操作更简便，是现有的表达谱检测手段的重要补充。NanostringnCounter 仪器一经问世大受欢迎，并已在 nature、science 、cell 等顶级杂志发表多篇文章，目前已将应用扩展至 miRNA、IncRNA、CNV 和融合基因的检测。我们会就一系列经典应用进行后续报道。Nanostring 近年来发表的文章2012 Passalacqua K.D. et al.,Strand-Specific RNA-Seq Reveals Ordered Patterns of Sense and Antisense Transcription
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