GSE62330
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. Overall design: Examine expression of miRNAs and piRNAs at testes of wild type and Mtdh deficent mice