Endogenous small RNAs of meristematic and a terminally differentiated tissue of rice.. Oryza sativa

To address the role of small regulatory RNAs in rice development, we generated a large data set of small RNAs from root apices (RoApx), shoot apices (ShApx), developing inflorescence (Infl) and mature leaf (Leaf). These tissues were chosen because they are expected to be rich in small RNAs and display different patterns of small RNA expression. The RoApx, ShApx, and Infl samples are expected to be enriched for meristematic tissues each of which have different fates. The Infl sample would also be expected to contain developing female gametes. In contrast, the Leaf sample is terminally differentiated and may be expected to be more transcriptionally repressed. These small RNAs comprise a total of 781,885 distinct sequences, and all of these map to at least one locus of the rice genome TIGR version 5. Overall design: Total RNA was extracted from root apices, shoot apices, developing inflorescence and mature leaf. Three samples from each tissue were taken to represent replicates and small RNAs from these were ligated to a common 5' RNA adapter and a replicate-specific 3' adapter. These replicate samples were then identified from within single 454 data sets using the 3' adapter sequence. It should be noted that there was a strong frequency bias observed between the RNA sequence and the particular 3' adapter used in the sequencing. This resulted in both increased or decreased read counts for the known miRNA sequences in a 3' adapter dependent fashion. Use of the three samples for each tissue as replicates is therefore problematic.

为阐明小型调控RNA（small regulatory RNAs）在水稻发育过程中的功能，我们构建了一套源自根尖（root apices, RoApx）、茎尖（shoot apices, ShApx）、发育中花序（developing inflorescence, Infl）及成熟叶片（mature leaf, Leaf）的大型小型RNA数据集。
选取上述组织的原因在于，其不仅富含小型调控RNA，且展现出各异的小型RNA表达模式。根尖、茎尖与发育中花序样本预期富集于具有不同分化命运的分生组织，其中发育中花序样本还会包含发育中的雌配子体。与之相对，成熟叶片样本已完成终末分化，转录抑制程度更高。
本次检测到的小型RNA共计781,885条独特序列，所有序列均可比对至水稻基因组TIGR版本5的至少一个基因组位点。
整体实验设计：从根尖、茎尖、发育中花序及成熟叶片中提取总RNA，每类组织设置三份生物学重复样本。将每份样本中的小型RNA连接通用5' RNA接头与重复特异性的3'接头，随后通过3'接头序列从单一454测序数据集中区分各重复样本。
需注意的是，测序所用的3'接头类型与RNA序列之间存在显著的频率偏倚，该偏倚会以依赖3'接头的方式导致已知微小RNA（microRNA, miRNA）序列的读长计数出现升高或降低。因此，将每类组织的三份样本作为生物学重复存在一定局限性。