Next Generation Sequencing Facilitates Quantitative Analysis of Altered Expression of miRNAs and piRNAs in the Testes of Mtdh exon 3-deficient Mice

Purpose: To identify the correlation of the expression of testicular miRNAs and piRNAs with the infertility of Mtdh exon 3 deficient mice Methods: Total RNAs from individual testis of two WT mice and three homozygous Mtdh exon 3-depleted mice were used to prepare the miRNA sequencing library. After the completed libraries were quantified with an Agilent 2100 Bioanalyzer, the DNA fragments in the libraries were denatured with 0.1M NaOH to generate single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ and sequenced for 36 cycles on Illumina HiSeq 2000 according to the manufacturer's instructions. Image analysis and base calling were performed using Off-Line Basecaller software (OLB V1.8.0). Subsequently, 3' adapter sequences were trimmed from clean reads (reads that passed Solexa CHASTITY quality filter) and any reads shorter than 15nt were discarded. Next the 3'-adapter-trimmed-reads (>= 15nt) were aligned to the latest known human reference miRNA precursor set (Sanger miRBase 19) using Novoalign (v2.07.11). Reads (counts < 2) were discarded when calculating miRNA expression. In order to characterize the isomiR variability, any sequences that matched the miRNA precursors in the mature miRNA region ±4nt (no more than one mismatch) were accepted as mature miRNA isomiRs, which were grouped according to the 5-prime (5p) or 3-prime (3p) arm of the precursor hairpin. For piRNAs, the 3'-adapter-trimmed reads (length >= 15nt) were aligned to the latest piRNA set in piRNAbank using Novoalign software (v2.07.11). Expression of each piRNA was defined as the mapped tag counts. Results: Reduced expression of miRNAs was detected in homozygous Mtdh exon 3-deficient testes (Table I). As shown in tables II and III, piRNA expression levels were dysregulated, with some being reduced and others increased in homozygous Mtdh exon 3-deficient testes compared to WT mice. Conclusions: Our study represents the first detailed analysis of Mtdh deficiency on the expression of small noncoding RNAs, generated by RNA-seq technology. We conclude that Mtdh is correlated to the expression of small non-coding RNAs including miRNAs and piRNAs at testis of mouse. Examine expression of miRNAs and piRNAs at testes of wild type and Mtdh deficent mice研究目的：本研究旨在明确睾丸微小RNA（miRNA）与piwi相互作用RNA（piRNA）的表达水平与Mtdh外显子3敲除小鼠不育症的相关性。 实验方法：提取2只野生型（WT）小鼠与3只纯合型Mtdh外显子3缺失小鼠的个体睾丸总RNA，用于构建miRNA测序文库。使用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）完成文库定量后，以0.1M氢氧化钠将文库中的DNA片段变性为单链DNA分子，随后将其结合于Illumina流动槽，经原位扩增后，严格按照厂商操作说明在Illumina HiSeq 2000测序平台上完成36个循环的测序。采用离线碱基识别软件（Off-Line Basecaller, OLB V1.8.0）完成图像分析与碱基识别。随后，对通过Solexa CHASTITY质量过滤的洁净读段去除3'接头序列，丢弃长度短于15nt的读段。将长度≥15nt的3'接头修剪后读段比对至最新版已知人类参考miRNA前体集（Sanger miRBase 19），比对工具为Novoalign（v2.07.11）。计算miRNA表达量时，丢弃读段计数<2的条目。为表征miRNA异构体（isomiR）的变异特征，将在成熟miRNA区域±4nt范围内（错配数不超过1个）匹配至miRNA前体的序列判定为成熟miRNA异构体，并根据前体发夹结构的5'臂（5p）或3'臂（3p）进行分组。针对piRNA分析：将经3'接头修剪且长度≥15nt的读段比对至piRNAbank数据库中的最新版piRNA集，比对工具同样为Novoalign（v2.07.11）。每个piRNA的表达量以比对上的标签计数定义。 实验结果：在纯合型Mtdh外显子3缺失小鼠的睾丸中，检测到miRNA表达水平下调（见表I）。如表II与表III所示，与野生型小鼠相比，纯合型Mtdh外显子3缺失小鼠睾丸中的piRNA表达失调，部分piRNA表达水平下调，部分则上调。 研究结论：本研究首次通过RNA测序技术，对Mtdh缺失导致的小型非编码RNA表达变化开展了详细分析。研究结果表明，Mtdh与小鼠睾丸内包括miRNA与piRNA在内的小型非编码RNA的表达水平密切相关。本研究考察了野生型与Mtdh缺陷小鼠睾丸中的miRNA与piRNA表达情况。