Novel transcripts discovered by mining genomic DNA from defined regions of bovine chromosome 6

Weikard, Rosemarie; Goldammer, Tom; Eberlein, Annett; Kühn, Christa

doi:10.1186/1471-2164-10-186

Cited by 13 publications

(9 citation statements)

References 54 publications

(54 reference statements)

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“…For each of the three cows, frozen samples (50 mg) of parenchymal tissue from the challenged and from a control (non-challenged) udder quarter were pulverized in liquid nitrogen, and total RNA was extracted with TRIzol reagent (Invitrogen, Darmstadt, Germany) followed by an on-column-purification using the NucleoSpin RNA II kit (Macherey & Nagel, Düren, Germany) with modifications of the DNase digestion step according to Weikard et al [39]. After testing the total RNA preparation for genomic DNA presence by PCR [40], the DNase-treatment step was repeated when necessary. The RNA concentration was measured with a Qubit Fluorometer (Invitrogen, Germany).…”

Section: Methodsmentioning

confidence: 99%

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue

et al. 2019

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Background In the mammary gland transcriptome of lactating dairy cows genes encoding milk proteins are highly abundant, which can impair the detection of lowly expressed transcripts and can bias the outcome in global transcriptome analyses. Therefore, the aim of this study was to develop and evaluate a method to deplete extremely highly expressed transcripts in mRNA from lactating mammary gland tissue. Results Selective RNA depletion was performed by hybridization of antisense oligonucleotides targeting genes encoding the caseins ( CSN1S1, CSN1S2, CSN2 and CSN3 ) and whey proteins ( LALBA and PAEP ) within total RNA followed by RNase H-mediated elimination of the respective transcripts. The effect of the RNA depletion procedure was monitored by RNA sequencing analysis comparing depleted and non-depleted RNA samples from Escherichia coli ( E. coli ) challenged and non-challenged udder tissue of lactating cows in a proof of principle experiment. Using RNase H-mediated RNA depletion, the ratio of highly abundant milk protein gene transcripts was reduced in all depleted samples by an average of more than 50% compared to the non-depleted samples. Furthermore, the sensitivity for discovering transcripts with marginal expression levels and transcripts not yet annotated was improved. Finally, the sensitivity to detect significantly differentially expressed transcripts between non-challenged and challenged udder tissue was increased without leading to an inadvertent bias in the pathogen challenge-associated biological signaling pathway patterns. Conclusions The implementation of selective RNase H-mediated RNA depletion of milk protein gene transcripts from the mammary gland transcriptome of lactating cows will be highly beneficial to establish comprehensive transcript catalogues of the tissue that better reflects its transcriptome complexity. Electronic supplementary material The online version of this article (10.1186/s12864-019-5781-3) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue

et al. 2019

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“…Genomic DNA was carefully eliminated from RNA preparations by repeated on-column digestion using twice the concentration of RNAse-free DNase I the manufacturers recommended in their protocols (Macherey & Nagel, Düren, Germany). Quality control of RNA was checked by a PCR specifically designed to detect genomic contamination [ 43 ]. RNA concentration and purity were measured using a NanoDrop ND-1000 spectrophotometer (peQLab, Erlangen, Germany).…”

Section: Methodsmentioning

confidence: 99%

Identification of novel transcripts and noncoding RNAs in bovine skin by deep next generation sequencing

2013

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BackgroundDeep RNA sequencing (RNAseq) has opened a new horizon for understanding global gene expression. The functional annotation of non-model mammalian genomes including bovines is still poor compared to that of human and mouse. This particularly applies to tissues without direct significance for milk and meat production, like skin, in spite of its multifunctional relevance for the individual. Thus, applying an RNAseq approach, we performed a whole transcriptome analysis of pigmented and nonpigmented bovine skin to describe the comprehensive transcript catalogue of this tissue.ResultsA total of 39,577 unique primary skin transcripts were mapped to the bovine reference genome assembly. The majority of the transcripts were mapped to known transcriptional units (65%). In addition to the reannotation of known genes, a substantial number (10,884) of unknown transcripts (UTs) were discovered, which had not previously been annotated. The classification of UTs was based on the prediction of their coding potential and comparative sequence analysis, subsequently followed by meticulous manual curation. The classification analysis and experimental validation of selected UTs confirmed that RNAseq data can be used to amend the annotation of known genes by providing evidence for additional exons, untranslated regions or splice variants, by approving genes predicted in silico and by identifying novel bovine loci. A large group of UTs (4,848) was predicted to potentially represent long noncoding RNA (lncRNA). Predominantly, potential lncRNAs mapped in intergenic chromosome regions (4,365) and therefore, were classified as potential intergenic lncRNA. Our analysis revealed that only about 6% of all UTs displayed interspecies conservation and discovered a variety of unknown transcripts without interspecies homology but specific expression in bovine skin.ConclusionsThe results of our study demonstrate a complex transcript pattern for bovine skin and suggest a possible functional relevance of novel transcripts, including lncRNA, in the modulation of pigmentation processes. The results also indicate that the comprehensive identification and annotation of unknown transcripts from whole transcriptome analysis using RNAseq data remains a tremendous future challenge.

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“…These evolutionary changes may be important for understanding metabolic adaptations to the ruminant lifestyle. Examination of defined regions of chromosome 6 revealed a number of novel transcripts [ 26 ] suggesting that the full complement of transcriptional activity of the genome is yet to be defined. This is a reminder of a major future challenge – the mapping of the complete repertoire of transcriptional activity across the genome.…”

Section: Commentarymentioning

confidence: 99%

Unlocking the bovine genome

et al. 2009

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Novel transcripts discovered by mining genomic DNA from defined regions of bovine chromosome 6

Cited by 13 publications

References 54 publications

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue

Identification of novel transcripts and noncoding RNAs in bovine skin by deep next generation sequencing

Unlocking the bovine genome

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