Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: 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

ORGANISM(S): Mus musculus

SUBMITTER: xiangbing meng

PROVIDER: E-GEOD-62330 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genetic Deficiency of Mtdh Gene in Mice Causes Male Infertility via Impaired Spermatogenesis and Alterations in the Expression of Small Non-coding RNAs.

Meng Xiangbing X Yang Shujie S Zhang Yuping Y Wang Xinjun X Goodfellow Renee X RX Jia Yichen Y Thiel Kristina W KW Reyes Henry D HD Yang Baoli B Leslie Kimberly K KK

The Journal of biological chemistry 20150318 19

Increased expression of metadherin (MTDH, also known as AEG-1 and 3D3/LYRIC) has been associated with drug resistance, metastasis, and angiogenesis in a variety of cancers. However, the specific mechanisms through which MTDH is involved in these processes remain unclear. To uncover these mechanisms, we generated Mtdh knock-out mice via a targeted disruption of exon 3. Homozygous Mtdh knock-out mice are viable, but males are infertile. The homozygous male mice present with massive loss of spermat ...[more]

PMID: 25787082

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Project description: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.

Project description:Purpose:To evaluate the effect of transient Global Cerebral Ischemia(tGCI) with Hypoxic Postconditioning (HPC) on piRNA expression profiles, piRNAs sequencing was performed. Methods: Total RNA of each sample (n=3 in each group) was prepared using Trizol reagent (Invitrogen), and the RNA quality and integrity were confirmed. 5’- and 3’-Adaptors were ligated to the obtained small RNA. Reverse transcription followed by PCR was used to create cDNA constructs. Subsequently, an about 150 bp fraction corresponding to approximate the adaptor-ligated constructs derived from the 30 nts piRNA fragment was excised and purified. The purified libraries were sequenced on Illumina HiSeq 2000 apparatus. The raw data were refined using cutadapt software (v1.9.1). After filtering out low quality reads and short reads (<15 nts), trimmed reads from all samples were pooled, and piano software was used to predict novel piRNAs. The trimmed reads were aligned to the merged rat piRNA databases (known piRNA from piRNABank plus the newly predicted piRNAs) using Novoalign software (v3.02.12) with at most one mismatch. PiRNA-binding transposon and targets were predicted by miranda software (v3.3a). piRNA-targets networks were plotted by cytoscape software (v2.8.0). Results: After tGCI, a total of 5574 piRNAs in CA1 was detected after 26 h of reperfusion. Of those, 12 were significantly upregulated compared with Sham rats (≥1.5-fold change, p<0.05), including 6 novel piRNAs. Of the 12 piRNAs, 4 showed ≥2-fold change. In comparison to tGCI group, 6 piRNAs in CA1 were significantly downregulated after HPC. Interestingly, the piRNAs rno_piR_000618, rno_piR_017990 and rno_piR_014971 were significantly changed in both tGCI and HPC groups Conclusions: In our study, we identified a cohort of piRNAs in CA1 of rats. Furthermore, we demonstrated directly contrast changes of piRNAs interacted with Piwil2 in CA1 after tGCI, as compared to those in HPC rats. Therefore, this study revealed that PIWI/piRNAs respond to ischemic brain damage and neuroprotection mediated by HPC. To the best of our knowledge, this is the first study ever on piRNAs associated with Piwil2 in cerebral ischemic tolerance.

Dataset Information

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

Publications

Genetic Deficiency of Mtdh Gene in Mice Causes Male Infertility via Impaired Spermatogenesis and Alterations in the Expression of Small Non-coding RNAs.

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