Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE88915 | GEO | 2017/02/22
SECONDARY ACCESSION(S): PRJNA349073
REPOSITORIES: GEO
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Project description:The ongoing ascent of sequencing technologies has enabled researchers to gain unprecedented insights into the RNA content of biological samples. MiRNAs, a class of small non-coding RNAs, play a pivotal role in regulating gene expression. The discovery that miRNAs are stably present in circulation has spiked interest in their potential use as minimally-invasive biomarkers. However, sequencing of blood-derived samples (serum, plasma) is challenging due to the often low RNA concentration, poor RNA quality and the presence of highly abundant RNAs that dominate sequencing libraries. In murine serum for example, the high abundance of tRNA-derived small RNAs called 5' tRNA halves hampers the detection of other small RNAs, like miRNAs. We therefore evaluated two complementary approaches for targeted depletion of 5' tRNA halves in murine serum samples. Using a protocol based on biotinylated DNA probes and streptavidin coated magnetic beads we were able to selectively deplete 95% of the targeted 5' tRNA half molecules. This allowed an unbiased enrichment of the miRNA fraction resulting in a 6-fold increase of mapped miRNA reads and 60% more unique miRNAs detected. Moreover, when comparing miRNA levels in tumor-carrying versus tumor-free mice, we observed a three-fold increase in differentially expressed miRNAs.
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Project description:Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples
| PRJNA349073 | ENA
Project description:BACKGROUND:RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification. RESULTS:EMBR-seq results in 90% of the sequenced RNA molecules from an E. coli culture deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq uses a single or a few oligonucleotides per rRNA, thereby making this new technology significantly more cost-effective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits for bacterial rRNA depletion, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA. CONCLUSIONS:EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples.
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Project description:RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species which are often present in low abundance. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification. EMBR-seq results in greater than 80% of the sequenced RNA molecules deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq employs a single oligonucleotide per rRNA, thereby making this new technology significantly more cost-effective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA. Thus, EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples.
2020-10-22 | GSE149666 | GEO
Project description:5' tRNA halves are abundantly present in small RNA libraries prepared from serum samples, thereby limiting the detection of other small RNA species. In this study, we developed and compared two protocols for the selective depletion of 5' tRNA halves in RNA isolated from murine serum samples. To evaluate the efficacy of both protocols on small RNA sequencing, we performed the two 5' tRNA halves depletion protocols on total RNA isolated from a serum pool collected from healthy mice. Each RNA sample was divided into three and the resulting fractions were either depleted using beads or RNase H, or used as a control. The experiment was performed in triplicate. Upon depletion, libraries were prepared using TruSeq Small RNA Library Preparation kit. For both protocols, very high depletion efficiencies were observed, with more than 99% of the targeted tRNA-types being depleted. This resulted in a more than 6-fold increase in mapped miRNA reads and 60% more detected mature miRNAs when performing small RNA sequencing on murine serum samples. Importantly, we observed no considerable effects on the relative expression values of miRNAs.
2017-02-22 | GSE88913 | GEO
Project description:Acute infections of bovine viral diarrhea virus (BVDV) lead to a range of clinical presentations. Laboratory tests for detection depend on collection of samples during a short viremia. Acutely infected animals remain largely undiagnosed. Transfer RNA halves (tsRNAs) are hypothesized to function like microRNAs to regulate gene expression during an immune response. The objective of this study was to identify tsRNAs in cattle that had been challenged with a non-cytopathic field strain of BVDV. Colostrum-deprived neonatal Holstein calves were either challenged with BVDV (n=5) or mock challenged (n=4). Sera was collected prior to challenge and days 4, 9, and 16 post challenge. RNA was extracted and read counts of small non-coding RNAs were assessed using next-generation sequencing. A total of 87,838,207 reads identified 41 different tsRNAs. Two 5' tsRNAs, tsRNAProAGG and tsRNAValAAC, differed across time. Two 5' tsRNAs, tsRNAGlyCCC and tsRNAGlyGCC, differed between treatment groups across time. Four days post challenge, 5' tsRNAGlyCCC and tsRNAGlyGCC were significantly lower in the challenged group than the control group. Further studies are needed to identify the importance and function of 5' tsRNAGlyCCC and tsRNAGlyGCC in serum samples of cattle challenged with BVDV.
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Project description:The tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs) are newly discovered noncoding RNAs in recent years. They are derived from specific cleavage of mature and pre-tRNAs and expressed in various cancers. They enhance cell proliferation and metastasis or inhibit cancer progression. Many studies have investigated their roles in the diagnosis, progression, metastasis, and prognosis of various cancers, but the mechanisms through which they are involved in resistance to cancer treatment are unclear. This review outlines the classification of tRFs and tiRNAs and their mechanisms in cancer drug resistance, thus providing new ideas for cancer treatment.
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Project description:Despite the precipitous decline in the cost of genome sequencing over the last few years, library preparation for RNA-seq is still laborious and expensive for high throughput screening for drug discovery. Limited availability of RNA generated by some experimental workflows poses an additional challenge and typically adds to the cost of RNA library preparation. In a search for low cost, automation-compatible RNA library preparation kits that also maintain strand specificity and are amenable to low input RNA quantities, we systematically tested two recent commercial technologies – Swift and Swift Rapid – using the Illumina TruSeq stranded mRNA, the de facto standard workflow for bulk transcriptomics, as our reference. We used the Universal Human Reference RNA (UHRR) (composed of equal quantities of total RNA from 10 human cancer cell lines) to benchmark differential gene expression in these kits, at input quantities ranging between 10 ng to 500 ng. Read quality and alignment metrics revealed high mapping efficiency and uniform read coverage through genes for all samples across all three kits. Normalized read counts between all treatment groups were in high agreement, with pairwise Pearson correlation coefficients >0.97. Compared to the Illumina TruSeq stranded mRNA kit, both Swift RNA library kits are cost effective and offer shorter workflow times enabled by their patented Adaptase technology. Furthermore, the Swift RNA kit allows for a relatively broader (and lower) input range, producing consistent results across diverse samples. The Swift Rapid RNA method is the fastest and most cost effective NGS workflow that is best suited for higher RNA yields, with the exact same RNA input range as the Illumina TruSeq kit. We also found the Swift RNA kit to produce the fewest number of differentially expressed genes and pathways attributable to input mRNA concentration.
2021-02-23 | GSE167300 | GEO
Project description:BACKGROUND:Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes severe encephalitis and respiratory disease with a high mortality rate in humans. During large outbreaks of the viral disease, serological testing of serum samples could be a useful diagnostic tool, which could provide information on not only the diagnosis of NiV disease but also the history of an individual with previous exposure to the virus, thereby supporting disease control. Therefore, an efficient method for the inactivation of NiV in serum samples is required for serological diagnosis. METHODS:We determined the optimal conditions for the inactivation of NiV infectivity in human serum using heating and UV treatment. The inactivation method comprised UV irradiation with a cover of aluminum foil for 30 min and heating at 56 °C for 30 min. RESULTS:With an optimized protocol for virus inactivation, NiV infectivity in serum samples (containing 6.0?×?105 TCID50) was completely inactivated. CONCLUSIONS:We developed a recommended protocol for the effective inactivation of NiV. This protocol would enable a regional or local laboratory to safely transport or process samples, including NiV, for serological testing in its biosafety level-2 facility.
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