Translational Paediatric Oncology, National Cancer Research Institute (IST), Largo R Benzi 10, Genoa, 16132, Italy.
BMC Genomics. 2010 Mar 3;11:149. doi: 10.1186/1471-2164-11-149.
Ultra Conserved Regions (UCRs) are a class of 481 noncoding sequences located in both intra- and inter-genic regions of the genome. The recent findings that they are significantly altered in adult chronic lymphocytic leukemias, carcinomas, and pediatric neuroblastomas lead to the hypothesis that UCRs may play a role in tumorigenesis.
We present a novel application of Ribo-SPIA isothermal linear amplification of minute RNA quantities for quantifying Transcribed-UCR (T-UCR) expression by quantitative PCR. Direct comparison of non-amplified with amplified cDNA in two neuroblastoma cell lines showed that the amplification approach increases sensitivity and repeatability in T-UCR quantification. It is noteworthy that the Ribo-SPIA step allowed us to analyze all 481 T-UCRs by using 150 ng of RNA, while introducing a minimal bias and preserving the magnitude of relative expression. Only the less abundant T-UCRs have high intra-assay variability, consistently with the Poisson distribution statistics and stochastic effects on PCR repeatability.
We demonstrated that the quantification procedure shown here is an accurate and reliable technique for genome-wide non coding gene (i.e., UCRs) profiling using small amounts of RNA. This issue is particularly important because studies of transcription regulation are increasingly conducted in small homogeneous samples, such as laser capture microdissected or sorted cell populations.
超保守区（UCRs）是一类位于基因组的内含子和外显子区域的 481 个非编码序列。最近的研究发现，它们在成人慢性淋巴细胞白血病、癌和小儿神经母细胞瘤中发生了显著改变，这使得人们提出假设，UCRs 可能在肿瘤发生中发挥作用。
我们提出了一种新的应用方法，即 Ribo-SPIA 等温线性扩增微量 RNA 数量，通过定量 PCR 来定量转录 UCR（T-UCR）的表达。在两种神经母细胞瘤细胞系中，将未扩增的 cDNA 与扩增的 cDNA 进行直接比较，结果表明扩增方法提高了 T-UCR 定量的灵敏度和可重复性。值得注意的是，Ribo-SPIA 步骤允许我们使用 150ng 的 RNA 分析所有 481 个 T-UCR，同时引入最小的偏差并保持相对表达的幅度。只有较少的 T-UCR 具有高的内实验变异性，这与泊松分布统计和 PCR 重复性的随机效应一致。
我们证明了这里所示的定量程序是一种使用少量 RNA 进行全基因组非编码基因（即 UCRs）分析的准确可靠的技术。这个问题特别重要，因为转录调控的研究越来越多地在小而同质的样本中进行，如激光捕获显微解剖或分选的细胞群体。
