荧光条形码标记的单分子检测技术.docx

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Nature 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，NanostringnCounter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 in Bacillus anthracis.PloS One. 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