Microarray probe design

MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Different probe designs were investigated with respect to sensitivity and selectivity of microarray hybridization. An array with 11 different variants of oligonucleotides for miR-122a and miR-16 was produced. 5 µg of respective total RNA was fluorescently labelled by 3' ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of 816 non redundant miRNAs sequences and miRControl 3 sequences. Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset).微小RNA（MicroRNAs, miRNAs）在诸多细胞过程、发育进程及生理活动中发挥着关键作用，已有大量研究证实了这一点。据此，学界已建立多种方法用于微小RNA的鉴定与定量分析。由于miRNA序列长度偏短，导致其解链温度的动态范围极宽，同时还会阻碍检测探针或优化后的聚合酶链式反应（PCR）引物的合理筛选。尽管miRNA微阵列可实现所有已知miRNA的大规模并行精准相对定量，但迄今为止，其在绝对定量检测方面的应用价值仍较为有限。本研究提出一种基于微阵列的微小RNA全局绝对定量分析方法。该方法依托已知浓度的约1000种合成miRNA的等摩尔混合池作为通用参考标准，通过双色标记与杂交策略，将其与待测样本共同在同一微阵列上进行杂交。每种单一miRNA均可通过与通用参考标准的比对实现定量，从而抵消由序列、标记、杂交或信号检测方法带来的系统偏差。本研究通过多组外参掺入实验验证了该方法的准确性。此外，我们还对肝脏样本以及CD34(+)CD133(-)造血干细胞中的miRNA拷贝数进行了定量分析。本研究针对微阵列杂交的灵敏度与特异性，对多种探针设计方案进行了考察。我们制备了针对miR-122a与miR-16的11种不同寡核苷酸变体的微阵列芯片。取5 μg对应总RNA，通过3'端连接法进行荧光标记。总RNA通过双色杂交策略与微阵列结合，同时与另一组标记好的合成miRNA混合池进行对照杂交。该合成miRNA混合池包含816种非冗余miRNA序列以及miRControl 3序列，每种组分的摩尔量均为5飞摩尔（fmol）。对信号值扣除局部背景以得到净信号强度；仅针对未被标记为无效的位点（空白位点、质量不佳位点、阴性位点），且待测样本来源的miRNA荧光强度为平均背景值两倍的位点，计算代表同一miRNA的4个对应位点的净信号强度平均值，该数据集记为2bkg数据集。