OBJECTIVE: Phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) signaling pathway is related to tumorigenesis by up-regulating survivin. Phosphatase and tensin homologue deleted on chromosome ten (PTEN) can suppress PI3K/AKT signaling pathway, while DJ-1 is the negative regulator of PTEN. DJ-1 up-regulation is closely correlated with the occurrence, progression, and drug resistance of pancreatic cancer. MicroRNA-142 (MiR-142) is significantly declined in pancreatic cancer tissue. Bioinformatics analysis demonstrated that complementary binding site exists between miR-142 and DJ-1. This investigation, therefore, aimed to study the role of miR-142 in the regulation of DJ-1-PTEN/PI3K/AKT/Survivin signaling pathway as well as in pancreatic cancer cell proliferation, apoptosis, and adriamycin (ADM) resistance.

MATERIALS AND METHODS: Dual luciferase assay was performed to assess the targeted relationship between miR-142 and DJ-1. MiR-142, DJ-1, and PTEN expressions in SW1990 cells and drug-resistant SW1990/ADM cells were compared. SW1990/ADM cells were divided into five groups, including mimic-NC, miR-142 mimic, small interfere normal control (si-NC), si-DJ-1, and miR-142 mimic + si-DJ-1 groups. DJ-1, PTEN, phosphorylated-AKT (p-AKT), and Survivin expressions were tested. Cell apoptosis was determined by flow cytometry. Cell proliferation was evaluated by EdU staining.

RESULTS: MiR-142 targeted inhibited DJ-1 expression. MiR-142, PTEN, and cell apoptosis significantly down-regulated, while DJ-1, p-AKT, Survivin, and cell proliferation significantly elevated in SW1990/ADM cells compared with SW1990 cells. MiR-142 mimics and/or si-DJ-1 transfection markedly reduced DJ-1, p-AKT, and Survivin expressions enhanced PTEN level, attenuated cell proliferation, enhanced cell apoptosis, and weakened ADM resistance.

CONCLUSIONS: MiR-142 over-expression weakened ADM resistance in pancreatic cancer cells by targeting DJ-1 to enhance PTEN expression and attenuate PI3K/AKT signaling pathway activity.

Features SPLIT RNA Extraction Kit

In this study, we used the channel catfish model clonal TS32.15 alloantigen-specific cytotoxic T cell (CTL) line to examine the dynamics of memory CTL expansion and senescence in teleosts. Although TS32.15 has been routinely cultured to study catfish CTL responses and killing mechanisms, little is known about the dynamics of the CTLs in these cultures. Here we show that this cell line consists of small non-cytotoxic T cells and larger granular effector T cells and that their ratios vary with time after stimulation. Small CTLs, when exposed to their irradiated targets, replicate and differentiate to morphologically distinct cytotoxic effectors, which do not replicate. After lysing target cells, or with prolonged absence of stimulation, the effector cells transition to a non-cytolytic senescent stage or become apoptotic. In addition, we demonstrate that natural IgM in catfish serum binds lipids, including PIP2, on early apoptotic CTLs, and that these IgM⁺ CTL can be cleared by catfish head kidney-derived macrophages.

Features SPLIT RNA Extraction Kit

Sporangia of Phytophthora infestans from pure cultures on agar plates are typically used in lab studies, whereas sporangia from leaflet lesions drive natural infections and epidemics. Multiple assays were performed to determine if sporangia from these two sources are equivalent. Sporangia from plate cultures showed much lower rates of indirect germination and produced much less disease in field and moist-chamber tests. This difference in aggressiveness was observed whether the sporangia had been previously incubated at 4 C (to induce indirect germination) or at 21 C (to prevent indirect germination). Furthermore, lesions caused by sporangia from plates produced much less sporulation. RNA-Seq analysis revealed that thousands of the >17,000 P. infestans genes with an RPKM >1 (reads per kilobase of exon model per million mapped reads >1) were differentially expressed in sporangia obtained from plate cultures of two independent field isolates compared to sporangia of those isolates from leaflet lesions. Among the significant differentially expressed genes (DEGs), putative RxLR effectors were over-represented, with almost half of the 355 effectors with RPKM>1 being up- or down-regulated. DEGs of both isolates included nine flagellar-associated genes, and all were down-regulated in plate sporangia. Ten elicitin genes were also detected as DEGs in both isolates, and nine (including INF1) were up-regulated in plate sporangia. These results corroborate previous observations that sporangia produced from plates and leaflets sometimes yield different experimental results and suggest hypotheses for potential mechanisms. We caution that use of plate sporangia in assays may not always produce results reflective of natural infections and epidemics.

Features SPLIT RNA Extraction Kit and SENSE mRNA-Seq Library Prep Kit

OBJECTIVE: The aberrant increasing expression of mammalian target of rapamycin (mTOR) participates in tumor occurrence and drug resistance. It has been found elevation of mTOR expression but reducing miR-107 expression in glioma tissues. Thus, we investigated the regulatory role of miR-107 on mTOR expression as well as glioma cell proliferation, apoptosis and cisplatin (DDP) resistance.

PATIENTS AND METHODS: Dual luciferase reporter gene assay was applied to confirm targeted regulation between miR-107 and mTOR. Tumor tissues were collected from glioma patients, in parallel with normal tissues after brain contusion surgery. Expressions of miR-107, mTOR and p-mTOR were compared. DDP-resistant cell line U251/DPP was generated. U251/DPP cells were further treated with miR-107 mimic or si-mTOR to examine the change of miR-107, mTOR, p-mTOR and survivin levels. Flow cytometry was used to quantify the effect of DDP treatment on cell proliferation or apoptosis.

RESULTS: Bioinformatics analysis revealed complementary binding sites between miR-107 and 3’-UTR of mTOR mRNA. Dual luciferase assay confirmed targeted regulation between miR-107 and mTOR. Compared to control group, in glioma tissues, mTOR and p-mTOR expressions were significantly elevated, while the level of miR-107 expression was markedly decreased. Of note, U251/DDP cells presented weakened apoptosis compared to U251 cells, with high levels of mTOR, p-mTOR and survivin and reduction of miR-107 expression. However, the transfection of miR-107 mimic and/or si-mTOR remarkably suppressed expressions of mTOR, p-mTOR and survivin in U251/DPP cells, weakened cell proliferation and enhanced apoptosis.

CONCLUSIONS: We demonstrated that the level of miR-107 was correlated with DDP resistance in glioma cells. Over-expression of miR-107 decreased DPP resistance of glioma cells via inhibition of mTOR, which provides academic basis for the future anti-glioma therapy.

Features SPLIT RNA Extraction Kit

Proper functioning of an organism requires cells and tissues to behave in uniform, well-organized ways. How this optimum of phenotypes is achieved during the development of vertebrates is unclear. Here, we carried out a multi-faceted and single-cell resolution screen of zebrafish embryonic blood vessels upon mutagenesis of single and multi-gene microRNA (miRNA) families. We found that embryos lacking particular miRNA-dependent signaling pathways develop a vascular trait similar to wild-type, but with a profound increase in phenotypic heterogeneity. Aberrant trait variance in miRNA mutant embryos uniquely sensitizes their vascular system to environmental perturbations. We discovered a previously unrecognized role for specific vertebrate miRNAs to protect tissue development against phenotypic variability. This discovery marks an important advance in our comprehension of how miRNAs function in the development of higher organisms.

Features SPLIT RNA Extraction Kit

The function, regulation and cellular distribution of GABA_A receptor subunits have been extensively documented in the adult rodent brain and are linked to numerous neurological disorders. However, there is a surprising lack of knowledge on the cellular (sub-) distribution of GABA_A receptor subunits and of their expressional regulation in developing healthy and diseased foetal human brains. To propose a role for GABA_A receptor subunits in neurodevelopmental disorders, we studied the developing hippocampus of normal and Down syndrome foetuses. Among the α1–3 and γ2 subunits probed, we find significantly altered expression profiles of the α1, α3 and γ2 subunits in developing Down syndrome hippocampi, with the α3 subunit being most affected. α3 subunits were selectively down-regulated in all hippocampal subfields and developmental periods tested in Down syndrome foetuses, presenting a developmental mismatch by their adult-like distribution in early foetal development. We hypothesized that increased levels of the amyloid precursor protein (APP), and particularly its neurotoxic β-amyloid (1–42) fragment, could disrupt α3 gene expression, likely by facilitating premature neuronal differentiation. Indeed, we find increased APP content in the hippocampi of the Down foetuses. In a corresponding cellular model, soluble β-amyloid (1–42) administered to cultured SH-SY5Y neuroblastoma cells, augmented by retinoic acid-induced differentiation towards a neuronal phenotype, displayed a reduction in α3 subunit levels. In sum, this study charts a comprehensive regional and subcellular map of key GABA_Areceptor subunits in identified neuronal populations in the hippocampus of healthy and Down syndrome foetuses and associates increased β-amyloid load with discordant down-regulation of α3 subunits.

Features SPLIT RNA Extraction Kit

OBJECTIVE: Over-proliferation of mesangial cells is the major pathological change of mesangial proliferative glomerulonephritis (MPGN). PTEN-PI3K/AKT pathway plays a role in regulating proliferation of mesangial cells. Anti-thymocyte serum nephritis (ATSN) is a widely used animal model for studying MPGN. This study established ATSN model, on which the role of PTEN-PI3K/AKT signal pathway in MPGN pathogenesis was investigated.

MATERIALS AND METHODS: ASTN rat model was established in parallel with control group. Protein expressions of PTEN, p-AKT, PCNA, Cyclin D1 and Bcl-2 were quantified, along with glomerular mesangial cell (GMC) counting. Rat mesangial cell (RMC) was treated with 0 or 10 ng/mL IL-6, followed by flow cytometry analysis for apoptosis, cycle and PCNA expression. Expressions of PTEN, p-AKT, PCNA, Cyclin D1 and Bcl-2 were measured. RMC was treated with pSicoR-PTEN and/or LY294002, followed by the treatment of 10 ng/mL IL-6 for 48 h. Cell apoptosis, cycle, PCNA expression and protein expression were measured.

RESULTS: Lower PTEN expression was found in renal cortex of ATSN rats, along with increasing levels of p-AKT, PCNA, Cyclin D1, Bcl-2, and higher GMCs, compared to that in control rats. IL-6 treatment increased protein expression in RMC, facilitated cell proliferation and cycle progression and suppressed apoptosis. Over-expression of PTEN and/or LY294002 remarkably decreased protein expression in RMC, inhibited the effect of IL-6 on proliferation, and induced cell apoptosis and cycle arrest.

CONCLUSIONS: The down-regulation of PTEN played a role in enhancing PI3K/AKT pathway activity, facilitating GMC proliferation and MPGN pathogenesis.

Features SPLIT RNA Extraction Kit

Estrogen is critical for skeletal homeostasis and regulates bone remodeling, in part, by modulating the expression of receptor activator of NF-κB ligand (RANKL), an essential cytokine for bone resorption by osteoclasts. RANKL can be produced by a variety of hematopoietic (e.g. T and B-cell) and mesenchymal (osteoblast lineage, chondrocyte) cell types. The cellular mechanisms by which estrogen acts on bone are still a matter of controversy. By using murine reconstitution models that allow for selective deletion of estrogen receptor-alpha (ERα) or selective inhibition of RANKL in hematopoietic vs. mesenchymal cells, in conjunction with in situ expression profiling in bone cells, we identified bone lining cells as important gatekeepers of estrogen-controlled bone resorption. Our data indicate that the increase in bone resorption observed in states of estrogen deficiency in mice is mainly caused by lack of ERα-mediated suppression of RANKL expression in bone lining cells.

Features SPLIT RNA Extraction Kit

MicroRNAs orchestrate brain functioning via interaction with microRNA recognition elements (MRE) on target transcripts. However, the global impact of potential competition on the microRNA pool between coding and non-coding brain transcripts that share MREs with them remains unexplored. Here we report that non-coding pseudogene transcripts carrying MREs (PSG^+MRE) often show duplicated origin, evolutionary conservation and higher expression in human temporal lobe neurons than comparable duplicated MRE-deficient pseudogenes (PSG^-MRE). PSG^+MRE participate in neuronal RNA-induced silencing complexes (RISC), indicating functional involvement. Furthermore, downregulation cell culture experiments validated bidirectional co-regulation of PSG^+MRE with MRE-sharing coding transcripts, frequently not their mother genes, and with targeted microRNAs; also, PSG^+MRE single-nucleotide polymorphisms associated with schizophrenia, bipolar disorder and autism, suggesting interaction with mental diseases. Our findings indicate functional roles of duplicated PSG^+MRE in brain development and cognition, supporting physiological impact of the reciprocal co-regulation of PSG^+MRE with MRE-sharing coding transcripts in human brain neurons.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina and SPLIT RNA Extraction Kit

RNA viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. Mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. New sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. Through selective host RNA depletion and compensatory protocol adjustments for ultra-low RNA inputs, we are able to detect three major blood-borne RNA viruses – HIV, HCV and HEV. We recovered complete genomes and up to 43% of the genome from samples with viral loads of 104 and 103 IU/ml respectively. Additionally, we demonstrated the utility of this method in detecting and characterising members of diverse RNA virus families within a human plasma background, some present at very low levels. By applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of HCV genotype 6, and a co-infecting human pegivirus. This method builds upon earlier RNA metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses.

Features SPLIT RNA Extraction Kit

Alzheimer’s disease (AD) involves changes in both lipid and RNA metabolism, but it remained unknown if these differences associate with AD’s cognition and/or post-mortem neuropathology indices. Here, we report RNA-sequencing evidence of inter-related associations between lipid processing, cognition level, and AD neuropathology. In two unrelated cohorts, we identified pathway-enriched facilitation of lipid processing and alternative splicing genes, including the neuronal-enriched NOVA1 and hnRNPA1. Specifically, this association emerged in temporal lobe tissue samples from donors where postmortem evidence demonstrated AD neuropathology, but who presented normal cognition proximate to death. The observed changes further associated with modified ATP synthesis and mitochondrial transcripts, indicating metabolic relevance; accordingly, mass-spectrometry-derived lipidomic profiles distinguished between individuals with and without cognitive impairment prior to death. In spite of the limited group sizes, tissues from persons with both cognitive impairment and AD pathology showed elevation in several drug-targeted genes of other brain, vascular and autoimmune disorders, accompanied by pathology-related increases in distinct lipid processing transcripts, and in the RNA metabolism genes hnRNPH2, TARDBP, CLP1 and EWSR1. To further detect 3′-polyadenylation variants, we employed multiple cDNA primer pairs. This identified variants that showed limited differences in scope and length between the tested cohorts, yet enabled superior clustering of demented and non-demented AD brains versus controls compared to total mRNA expression values. Our findings indicate inter-related cognition-associated differences in AD’s lipid processing, alternative splicing and 3′-polyadenylation, calling for pursuing the underlying psychological and therapeutics implications.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina and SPLIT RNA Extraction Kit

To assess the impact growth substrate has on the resulting sporangia, field and laboratory based inoculation experiments were carried out using three isolates of P. infestans clonal lineage US-23. Isolates were grown in pure culture on agar plates as well as on detached Solanum lycopersicum leaflets in moist chambers. Sporangia harvested from each of these culture types and for each isolate was used as inoculum in two independent field trials and one laboratory based trial in the summer of 2015. Disease assessment at 144 hours after inoculation was very consistent across all trials. In every case, culture derived sporangia produced significantly less disease than sporangia of the same isolate washed from sporulating leaflets. Genes associated with necrotrophy and host cell death were up regulated in culture grown sporangia, while genes associated with biotrophic growth and disease were more up regulated in sporangia from leaflets.

Features SENSE mRNA‐Seq Library Prep Kit
Features SPLIT RNA Extraction Kit

Analysis of fusion transcripts has become increasingly important due to their link with cancer development. Since high-throughput sequencing approaches survey fusion events exhaustively, several computational methods for the detection of gene fusions from RNA-seq data have been developed. This kind of analysis, however, is complicated by native trans-splicing events, the splicing-induced complexity of the transcriptome and biases and artefacts introduced in experiments and data analysis. There are a number of tools available for the detection of fusions from RNA-seq data; however, certain differences in specificity and sensitivity between commonly used approaches have been found. The ability to detect gene fusions of different types, including isoform fusions and fusions involving non-coding regions, has not been thoroughly studied yet. Here, we propose a novel computational toolkit called InFusion for fusion gene detection from RNA-seq data. InFusion introduces several unique features, such as discovery of fusions involving intergenic regions, and detection of anti-sense transcription in chimeric RNAs based on strand-specificity. Our approach demonstrates superior detection accuracy on simulated data and several public RNA-seq datasets. This improved performance was also evident when evaluating data from RNA deep-sequencing of two well-established prostate cancer cell lines. InFusion identified 26 novel fusion events that were validated in vitro, including alternatively spliced gene fusion isoforms and chimeric transcripts that include intergenic regions. The toolkit is freely available to download from http:/bitbucket.org/kokonech/infusion.

Features SENSE mRNA‐Seq Library Prep Kit
Features SPLIT RNA Extraction Kit

Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in “DNA damage response”, “direct p53 effectors” and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols.

Features QuantSeq 3′ mRNA-Seq Library Prep Kits
Features SPLIT RNA Extraction Kit

WHRN (DFNB31) mutations cause diverse hearing disorders: profound deafness (DFNB31) or variable hearing loss in Usher syndrome type II. The known role of WHRN in stereocilia elongation does not explain these different pathophysiologies. Using spontaneous and targeted Whrn mutants, we show that the major long (WHRN-L) and short (WHRN-S) isoforms of WHRN have distinct localizations within stereocilia and also across hair cell types. Lack of both isoforms causes abnormally short stereocilia and profound deafness and vestibular dysfunction. WHRN-S expression, however, is sufficient to maintain stereocilia bundle morphology and function in a subset of hair cells, resulting in some auditory response and no overt vestibular dysfunction. WHRN-S interacts with EPS8, and both are required at stereocilia tips for normal length regulation. WHRN-L localizes midway along the shorter stereocilia, at the level of inter-stereociliary links. We propose that differential isoform expression underlies the variable auditory and vestibular phenotypes associated with WHRN mutations.

Features SPLIT RNA Extraction Kit

X-linked hypophosphatemia (XLH) is the most frequent form of inherited rickets in humans caused by mutations in the phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX). Hyp mice, a murine homologue of XLH, are characterized by hypophosphatemia, inappropriately low serum vitamin D levels, increased serum fibroblast growth factor-23 (Fgf23), and osteomalacia. Although Fgf23 is known to be responsible for hypophosphatemia and reduced vitamin D hormone levels in Hyp mice, its putative role as an auto-/paracrine osteomalacia-causing factor has not been explored. We recently reported that Fgf23 is a suppressor of tissue nonspecific alkaline phosphatase (Tnap) transcription via FGF receptor-3 (FGFR3) signaling, leading to inhibition of mineralization through accumulation of the TNAP substrate pyrophosphate. Here, we report that the pyrophosphate concentration is increased in Hyp bones, and that Tnap expression is decreased in Hyp-derived osteocyte-like cells but not in Hyp-derived osteoblasts ex vivo and in vitro. In situ mRNA expression profiling in bone cryosections revealed a ~70-fold up-regulation of Fgfr3 mRNA in osteocytes versus osteoblasts of Hyp mice. In addition, we show that blocking of increased Fgf23-FGFR3 signaling with anti-Fgf23 antibodies or an FGFR3 inhibitor partially restored the suppression of Tnap expression, phosphate production, and mineralization, and decreased pyrophosphate concentration in Hyp-derived osteocyte-like cells in vitro. In vivo, bone-specific deletion of Fgf23 in Hyp mice rescued the suppressed TNAP activity in osteocytes of Hyp mice. Moreover, treatment of wild-type osteoblasts or mice with recombinant FGF23 suppressed Tnap mRNA expression and increased pyrophosphate concentrations in the culture medium and in bone, respectively. In conclusion, we found that the cell autonomous increase in Fgf23 secretion in Hyp osteocytes drives the accumulation of pyrophosphate through auto-/paracrine suppression of TNAP. Hence, we have identified a novel mechanism contributing to the mineralization defect in Hyp mice.

Features SPLIT RNA Extraction Kit

Although parathyroid hormone (PTH) has long been known to act as a bone anabolic agent when administered intermittently, the exact underlying mechanisms remain largely unknown. Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, has been identified to be a PTH target gene in vitro and in vivo. Here, we used female global AREG knockout (AREG-KO) mice to explore the role of AREG in mediating the bone anabolic effects of PTH. AREG-KO mice were characterized by unchanged distal femoral cancellous bone mass and only subtle decreases in bone mineral density (BMD) and cortical thickness at the femoral midshaft at 3 and 8 months of age, relative to wildtype controls. AREG deficiency was associated with complex changes in the mRNA expression of other EGFR ligands in femoral cancellous bone osteoblasts in situ in 3-week-old mice. To examine the bone anabolic effects of PTH in the absence and presence of AREG, we injected 3-month-old AREG-KO females and wildtype control littermates with 80 μg/kg PTH or vehicle 5 times per week over 4 weeks. Intermittent PTH treatment of AREG-KO mice led to increases in femoral trabecular and cortical BMD, cortical thickness, endocortical and periosteal bone formation, cancellous bone formation rate, and serum osteocalcin, comparable to those observed in wildtype control mice. In conclusion, our data indicate that the bone anabolic effects of PTH do not require AREG, at least in 3-month-old female mice.

Features SPLIT RNA Extraction Kit

Expression of E-cadherin has a central role in maintaining epithelial morphology. In solid tumors, reduction of E-cadherin results in disruption of intercellular contacts. Consequently, cells lose adhesive properties and gain more invasive mesenchymal properties. Nevertheless, the mechanism of E-cadherin regulation is not completely elucidated. Here we analyzed the distribution of E-cadherin expression at the cell level in humanhepatocellular carcinoma, in which human liver paraffin blocks from 25 hepatocellular carcinoma patients were prepared from cancerous (CA) and noncancerous areas (NCA). In situ hybridization (ISH) was performed to detect E-cadherin and hypoxia-induced factor-1α (HIF1α) mRNAs and immunohistochemistry to stain E-cadherin protein. In parallel, RNA was extracted from CA and NCA, and E-cadherin and HIF1α were quantified by quantitative reverse transcription PCR. ISH revealed abundant E-cadherin mRNA in nuclei of hepatocellular carcinoma cells (HCCs), whereas immunohistochemistry showed depletion of E-cadherin protein from these areas. In sections of NCA, E-cadherin mRNA was also found in the cytosol, and E-cadherin protein was detected on the membrane of cells. Experiments in cell lines confirmed E-cadherin mRNA in nuclei of cells negative for E-cadherin protein. HIF1α expression is elevated in CAs, which is associated with a clear cytosolic staining for this mRNA. Our results demonstrate that E-caderhin mRNA is selectively retained in nuclei of HCCs, whereas other mRNAs are still exported, suggesting that translocation of E-cadherinmRNA from nuclei to cytoplasm has a role in regulating E-cadherin protein levels during epithelial mesenchymal transition.

Features SPLIT RNA Extraction Kit