定量PCR检测microRNA表达的数据归一化新技术

A Novel Normalization Approach for MicroRNA Quantitative PCR

数据归一化技术对研究结果的分析具有重要的作用.在定量PCR实验中,通常利用稳定表达的看家基因作为实验数据归一化的内参,但最近的研究表明,这些看家基因的表达量在不同的生理病理过程中也表现出显著的变化,不适合作为数据归一化处理的基准.针对这一问题,提出一种新的数据处理技术,利用单细胞归一化方法(percellome),对定量PCR检测miRNA表达的数据进行处理,显著提高了数据处理的准确性.以8周龄/40周龄小鼠脑为实验材料,选择14种microRNA的表达情况进行了检测.在研究中,将3种不同拷贝数的人工合成的RNA片段(spike RNA)作为内参加入到样品中,用于microRNA表达检测的归一化基准.研究发现,未经处理的microRNA单细胞拷贝数的变化范围为2.0×105～4.3×105,而经单细胞归一处理,上述表达变化范围为2到26倍.该项研究还发现,看家基因U6 ncRNA和5S rRNA的表达水平存在显著的变化,在以基因组DNA为归一化基准时,其表达量变化为1.5和4.8倍,而在以RNA水平为归一化基准时,其表达量变化为5.8和3.8倍,表明这些基因不适合作为数据处理的基准.据此,为microRNA的定量研究提供了一种新的、可靠的归一化方案.

Data normalization plays a crucial role in the interpretation of experimental result.House-keeping genes were utilized as internal controls to accurately determine the gene expression in quantitative PCR.However,significant expression variation of these internal controls was revealed recently.A novel normalization approach（per cell normalization,percellome）,which is based on DNA and RNA normalization,is developed to calibrate miRNA expression in quantitative PCR.In which,a cocktail of three external RNAs with different copy numbers were spiked,so as be able to normalize miRNA expression against cell number.Gene expression of 14 miRNAs,as well as commonly used internal controls（U6 ncRNA and 5S rRNA）,were examined in the brain samples of 8 and 40 week-old mice.By using ＂per cell normalization＂ method,the expression level of theses miRNAs varied from 2-to 26-fold,while the absolute miRNA copy number per cell were from 2.0×105 to 4.3×105 copies per cell.Interestingly,the fold-change of U6 ncRNA and 5S rRNA were found to be 1.5-and 4.8-fold（based on DNA normalization）,and 5.8-and 3.8-fold（based on RNA normalization）,indicating significant expression variations of these two house-keeping genes.The study provides an novel approach to reliably normalize miRNA expression in quantitative PCR.