MicroRNA expression in the newborn mouse cochlea and vestibule

MicroRNAs (miRNAs) inhibit the translation of target mRNAs and affect, directly or indirectly, the expression of a large portion of the protein-coding genes. This study focuses on miRNAs that are expressed in the mouse cochlea and vestibule, the two inner ear compartments. To identify miRNAs that are expressed in the vertebrate developing inner ear, we used miRNA microarrays. Similar miRNA profiles were found in newborn (P0) mouse whole cochleae and vestibules. 105 miRNAs were found to be expressed in the whole P0 cochlea and 114 miRNAs expressed in the whole P0 vestibule with average intensities higher than twice the global background, out of 206 included in the arrays. Only 24 miRNAs were found to have different levels of expression in these whole organs, and these differences were mild (15-40%). The microarray results were intersected with two bioinformatic complementary approaches in order to choose candidate miRNAs that are predicted to be expressed specifically in the inner ear sensory epithelia (see the paper). Six small (<40 nt) RNA samples were hybridized with 4 microarrays (to save costs, some of the microarrays were hybridized with two different samples, labeled with different CyDyes – Cy3 and Cy5). Each RNA sample contains small RNAs (<40 nt) pooled from at least 20 newborn cochleae or vestibules, and there are 3 different samples for each organ. However, one of the samples (WT_P0_whole_Cochlea_3_V4) was hybridized with a different array than the other samples (printed from the same probes, but at a different date), and therefore excluded from the analysis described in the paper. Each microarray contains at least 4 identical spots per each probe that may be considered as technical replicates. Four of the samples were hybridized in parallel at the same day to two microarrays, and the dyes were swaped: First microarray: WT_P0_whole_Cochlea_1 (Cy3) [GSM388075] and WT_P0_whole_Vestibule_2 (Cy5) [GSM388635]; second microarray: WT_P0_whole_Cochlea_2 (Cy5) [GSM388633] and WT_P0_whole_Vestibule_1 (Cy3) [GSM388634]. The other 2 samples: WT_P0_whole_Vestibule_3 (Cy3) [GSM388637] and WT_P0_whole_Cochlea_3_V4 (Cy3) [GSM388660] were hybridized to two individual microarrays, at different dates. The miRNA profile in the cochlear and vestibular samples were compared: the vestibular samples are considered as the “experiment” samples, and the cochlear samples – as “reference”. Positive control probes (labeled as “control 1” in raw data) were added to RNA samples before labeling. The average of empty and buffer spots was used to calculate the global background of each array, and only miRNAs with an average expression higher than twice the fold of the array global background at least in one of the tissues were considered as expressed.微小RNA（microRNAs，miRNAs）可抑制靶信使RNA（messenger RNA，mRNA）的翻译过程，并直接或间接影响绝大多数编码蛋白基因的表达水平。本研究聚焦于在小鼠耳蜗与前庭这两种内耳分区中表达的微小RNA。 为鉴定在脊椎动物发育内耳中表达的微小RNA，本研究采用了微小RNA芯片（miRNA microarray）技术。新生（出生后第0天，P0）小鼠的全耳蜗与全前庭样本中，检测到了相似的微小RNA表达谱。在所检测的206个芯片探针中，全P0耳蜗样本内有105个微小RNA得以检出，全P0前庭样本内则有114个微小RNA的平均信号强度高于全局背景值的两倍。仅24个微小RNA在这两种全器官样本中呈现表达差异，且差异幅度较小（15%~40%）。 为筛选出预测可在内耳感觉上皮中特异性表达的候选微小RNA，本研究将芯片结果与两种互补的生物信息学分析方法进行了交叉验证（详见原文）。 本研究共制备6份小分子RNA（<40 nt）样本，与4张芯片进行杂交实验：为节约成本，部分芯片可同时与两份采用不同荧光染料（Cy3与Cy5）标记的样本进行杂交。每份RNA样本均由至少20份新生小鼠耳蜗或前庭样本中的小分子RNA混合制备而成，每种器官对应3份独立样本。但其中一份样本（WT_P0_whole_Cochlea_3_V4）采用了与其余样本不同的芯片进行杂交（该芯片使用相同探针，但制备日期不同），因此被排除于本文所述的分析流程之外。 每张芯片上每个探针至少设置4个重复点样，可作为技术重复样本。其中4份样本于同日平行与两张芯片进行杂交，并进行了荧光染料交换：第一张芯片：WT_P0_whole_Cochlea_1（Cy3）[GSM388075] 与 WT_P0_whole_Vestibule_2（Cy5）[GSM388635]；第二张芯片：WT_P0_whole_Cochlea_2（Cy5）[GSM388633] 与 WT_P0_whole_Vestibule_1（Cy3）[GSM388634]。剩余两份样本——WT_P0_whole_Vestibule_3（Cy3）[GSM388637] 与 WT_P0_whole_Cochlea_3_V4（Cy3）[GSM388660]——分别于不同日期与两张独立芯片进行杂交。 本研究对耳蜗与前庭样本的微小RNA表达谱进行了比较分析：前庭样本设为"实验"组样本，耳蜗样本则设为"参照"组样本。 在标记步骤前，已向所有RNA样本中加入阳性对照探针（原始数据中标记为"control 1"）。 本研究采用空白点与缓冲液点的平均信号值计算每张芯片的全局背景值，仅将至少在一种组织中平均表达量高于芯片全局背景值两倍的微小RNA认定为已表达。