Relative quantification of miRNAs across multiple experiments

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. We analyzed to which extend the universal reference can be used as a tool for the relative quantification of miRNAs across multiple experiments. We compared the results of direct hybridizations i.e. sample vs. sample to those of indirect hybridizations i.e. sample vs. UR. For the direct hybridizations, we hybridized 5µg liver total RNA vs 5 µg brain total RNA (n = 3) and for the indirect hybridization 5 µg liver or brain total RNA vs UR (5 fmol/miRNA) (n = 3). We calculated the so-called re-ratios for the UR experiments by dividing the signal ratios of the liver vs. UR array by the respective brain vs. UR array gaining a liver vs. brain re-ratio. Each RNA sample was mixed with 5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled. The RNA mix was hybridized in a dual colour approach to microarrays. The mean ratios of all probes were normalized to the median of the ratios detected for the spiked 18 synthetic RNA oligonucleotides reverse complement to miRControl 3 probes.已有研究证实，微RNA（microRNAs, miRNAs）可在诸多细胞、发育及生理过程中发挥关键调控作用。因此，学界已建立多种方法用于miRNA的鉴定与定量分析。但miRNA序列较短，导致其解链温度动态范围跨度较大，同时也阻碍了检测探针或优化型PCR引物的合理筛选。尽管miRNA芯片可对所有已知miRNA实现大规模并行且精准的相对定量，但截至目前，其在绝对定量检测领域的应用价值仍较为有限。本研究提出一种基于芯片的方法，可实现miRNA的全域绝对定量。该方法依托包含约1000种已知浓度合成miRNA的等摩尔混合池作为通用参考品（universal reference, UR），采用双色标记杂交策略，将其与待测样本一同在同一芯片上完成杂交。通过将每个单一miRNA的定量结果与通用参考品进行比对，可有效抵消由序列、标记、杂交或信号检测方法带来的系统偏差。本研究通过多组加标实验验证了该方法的准确性。此外，我们还对肝脏样本及CD34(+)CD133(-)造血干细胞中的miRNA拷贝数进行了定量分析。我们还分析了该通用参考品可在多大程度上作为跨实验场景的miRNA相对定量工具。我们分别比对了直接杂交（即样本与样本间的杂交）与间接杂交（即样本与通用参考品（UR）间的杂交）的实验结果。直接杂交组中，我们采用5μg肝脏总RNA与5μg脑组织总RNA进行杂交（n=3）；间接杂交组则采用5μg肝脏或脑组织总RNA与UR（每种miRNA 5fmol）进行杂交（n=3）。我们通过将肝脏与UR芯片的信号比值除以对应脑组织与UR芯片的信号比值，计算得到所谓的‘重比值（re-ratios）’，进而获得肝脏与脑组织间的重比值。每个RNA样本均与18种与miRControl 3探针反向互补的RNA寡核苷酸（每种5fmol）混合，随后进行荧光标记。将混合后的RNA采用双色杂交策略与芯片完成杂交。我们将所有探针的平均比值以加标的18种与miRControl 3探针反向互补的合成RNA寡核苷酸的检测比值中位数进行归一化处理。