RNase Protection Assay Research Articles

Upregulation of functional Kv11.1a isoform expression by modified U1 small nuclear RNA

The KCNH2 or human ether-a go-go-related gene (hERG) encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier potassium current in the heart. The expression of Kv11.1 C-terminal isoforms is directed by the alternative splicing and polyadenylation of intron 9. Splicing of intron 9 leads to the formation of a functional, full-length Kv11.1a isoform and polyadenylation of intron 9 results in the production of a non-functional, C-terminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO plays an important role in regulating Kv11.1 channel function. In the heart, only one-third of KCNH2 pre-mRNA is processed to Kv11.1a due to the weak 5′ splice site of intron 9. We previously showed that the weak 5′ splice site is caused by sequence deviation from the consensus, and that mutations toward the consensus sequence increased the efficiency of intron 9 splicing. It is well established that 5′ splice sites are recognized by complementary base-paring with U1 small nuclear RNA (U1 snRNA). In this study, we modified the sequence of U1 snRNA to increase its complementarity to the 5′ splice site of KCNH2 intron 9 and observed a significant increase in the efficiency of intron 9 splicing. RNase protection assay and western blot analysis showed that modified U1 snRNA increased the expression of the functional Kv11.1a isoform and concomitantly decreased the expression of the non-functional Kv11.1a-USO isoform. In patch-clamp experiments, modified U1 snRNA significantly increased Kv11.1 current. Our findings suggest that relative expression of Kv11.1 C-terminal isoforms can be regulated by modified U1 snRNA.

1505P - Analysis of expression of immunomodulation factors in alveolar soft-part sarcoma: a retrospective study from the Spanish Group for Research on Sarcoma (GEIS)

Background: Alveolar soft-part sarcoma (ASPS) is an exceedingly rare entity. We previously reported on a series of 12 ASPS the expression of PD-1/PD-L1 and lymphocytic infiltrates. PD-L1 staining was positive in 50% of the cases, suggesting a role for immunomodulatory interventions. We now enlarge the series and explore the expression of immune-related genes with the HTG EdgeSeq System and analyze their possible prognostic role. Methods: Patients (pts) diagnosed with ASPS from Dec 1994 to Jul 2016 were reviewed. Clinical characteristics and outcome were collected. Immunohistochemical expression was tested on archived Formalin-Fixed Paraffin-Embedded (FFPE) blocks using PD-L1 (ab205921; Abcam), CD8 (ab4055; Abcam) antibodies. An ImmunOncology (IO) panel of 549 mRNAs was evaluated with the HTG EdgeSeq System from one 5µm FFPE section. This novel technology consists in a tissue digestion followed by a pre-hybridization with specific probes using quantitative nuclease protection assay (qNPA) and quantified by a standard RNA-seq protocol in a NGS sequencer. Results: We identified 16 ASPS pts: median age 23y (6-62), M/F=5/11. Stage localized/metastatic in 11/5. With a median FU of 91 mos (4-151), 6/11 pts (54%) relapsed, with a median RFS of 19 mos (95% CI 1-75) and 2 pts died. PD-L1 was pos in tumor in 10/16 (62%) pts. HTG showed differential expression of immune-related genes according to stage (localized vs metastatic) in MNDA (log2 fold change: -3.01, p < 0.00001) and PRAME (log2 fold change: 1.9, p < 0.0002). Pts relapsing differentially expressed ABCC2 (log2 fold change -1,64, p < 0.04) when compared with those not relapsing. PD-L1 pos vs neg tumors (pos>5% membrane staining for PD-L1) differentially expressed TNFSF11 (fold change: -1,77, p = 0.05). Tumors infiltrated/not for CD8 lymphs (pos>10% of CD8) had a differential expression of CCL18 (log2 fold change: 4,89, p < 0.0001) and SLAMF7 (log2 fold change 2,37, p < 0.0089). Conclusions: PD-L1 is expressed in more than a half of ASPS of our series. Differential expression in immune-associated genes suggest an immune related gene profile on this entity with clinical and pathological implications. Further exploration of immune-modulation in ASPS is ongoing. Legal entity responsible for the study: Spanish Group for Research on Sarcoma Funding: Spanish Group for Research on Sarcoma (GEIS). Disclosure: All authors have declared no conflicts of interest.

Abstract 174: Regulation of Kv11.1 C-terminal Isoform Expression by Polypyrimidine Tract Binding Protein

The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative processing. Intron 9 splicing leads to the formation of a functional, full-length Kv11.1a isoform, and polyadenylation within intron 9 generates a non-functional, C-terminally truncated Kv11.1a-USO isoform. In this study we investigated the developmental regulation of Kv11.1 isoform expression. We showed that Kv11.1a expression was lower than that of Kv11.1a-USO in the adult heart, but the levels of Kv11.1a and Kv11.1a-USO were similar in the fetal heart. We studied the effect of polypyrimidine tract binding protein (PTB) on the alternative processing of KCNH2 pre-mRNA. PTB is an RNA-binding protein well known for its role in the regulation of alternative splicing. Recently, PTB has been shown to regulate polyadenylation. We showed that PTB increased Kv11.1a isoform expression and decreased Kv11.1a-USO isoform expression by the RNase protection assay and immunoblot analysis. In patch-clamp experiments, we found that PTB significantly increased Kv11.1 current. Our findings suggest that the relative expression of Kv11.1 C-terminal isoforms can be regulated by PTB. It has been reported that PTB protein abundance is progressively reduced during postnatal heart development. Thus, PTB may play an important role in developmental regulation of Kv11.1 isoform expression in the heart.

Purification and characterization of adenovirus core protein VII: a histone-like protein that is critical for adenovirus core formation.

Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98 %, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

Abstract 5348: Reliable gene expression profiling from smaller and H&amp;E stained clinical FFPE specimens using EdgeSeq platform

Abstract Background: The scarcity of biological specimens and suboptimal quality of collected material, make clinical biomarker studies challenging. The EdgeSeq system, a novel genomics analysis platform which combines quantitative nuclease protection assay (qNPA) technology with next-generation sequencing (NGS), has the potential to mitigate the aforementioned challenges. The technology allows for a limited sample input and delivers high quality gene expression profiling data from challenging material such as FFPE tissues. However, there is further unmet need toward even smaller tissue input or in taking advantage of archival stained FFPE slides to fill the data gap, specifically for studying tumor microenvironment in the oncology clinical trials Methods: We performed EdgeSeq analysis on six triplets of unstained, H&amp;E stained and IHC stained slides from non-small cell lung cancer (NSCLC) patients tumor tissue using Oncology biomarker panel (OBP). Next, we extended the EdgeSeq OBP analysis to 44 pairs of unstained and H&amp;E stained slides from NSCLC to confirm the good correlation. To assess feasibility of limited tissue input, we first optimized bead-to-DNA volume ratio during NGS library cleanup by testing 4 ratios of bead-to-DNA from 2.5 to 1.0. Next, crude lysate prepared from small cell lung cancer (SCLC) FFPE tissue was 2-fold diluted from equivalent tissue area of 5mm2 to 0.08 mm2, and was subjected to EdgeSeq OBP profiling using the optimal bead-to-DNA ratio. Finally, correlation of gene expression profiles between stained and unstained pairs were calculated. Results: Within the six triplets of unstained, H&amp;E stained and IHC stained slides, the gene expression profiles from H&amp;E slides showed good correlation with the profiles from matched unstained FFPE. An extended NSCLC sample set of 44 pairs of unstained and H&amp;E stained FFPE further confirmed overall good correlation with 84% (37 out of 44 ) pairs with Pearson correlation coefficients of 0.9 or higher. In assessing the limited tissue input, crude lysate was diluted to as little as equivalent tissue area of 0.08mm2. The tissue area as small as 0.31 mm2 resulted in Pearson correlation coefficients of 0.9 with 5mm2 surface area input. Conclusion: We demonstrated feasibility of using EdgeSeq to generate reliable gene expression data from H&amp;E stained FFPE tissues. In addition, we showed that EdgeSeq platform can be used to generate reliable expression data from crude FFPE tissue lysate equivalent to surface area as low as 0.31 mm2 (size of larger laser microdissection tile or half of a tissue microarray core). The data show that EdgeSeq can be considered as a platform of choice to analyze specimens of suboptimal quality or of limited quantity and thus may help to fill the biomarker data gap in the oncology clinical trials, including enabling research into patterns of interactions and mechanisms of response and resistance of immune cell engagement with tumor at the loco-regional level. Citation Format: Zhenhao Qi, Lisu Wang, Keyur Desai, John Cogswell, Mark Stern, Byron Lawson, Bonnie LaFleur, Patrik Vitazka. Reliable gene expression profiling from smaller and H&amp;E stained clinical FFPE specimens using EdgeSeq platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5348. doi:10.1158/1538-7445.AM2017-5348

Discrimination of candidate subgenome-specific loci by linkage map construction with an S1 population of octoploid strawberry (Fragaria\u2009\xd7\u2009ananassa)

BackgroundThe strawberry, Fragaria × ananassa, is an allo-octoploid (2n = 8x = 56) and outcrossing species. Although it is the most widely consumed berry crop in the world, its complex genome structure has hindered its genetic and genomic analysis, and thus discrimination of subgenome-specific loci among the homoeologous chromosomes is needed. In the present study, we identified candidate subgenome-specific single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) loci, and constructed a linkage map using an S1 mapping population of the cultivar ‘Reikou’ with an IStraw90 Axiom® SNP array and previously published SSR markers.ResultsThe ‘Reikou’ linkage map consisted of 11,574 loci (11,002 SNPs and 572 SSR loci) spanning 2816.5 cM of 31 linkage groups. The 11,574 loci were located on 4738 unique positions (bin) on the linkage map. Of the mapped loci, 8999 (8588 SNPs and 411 SSR loci) showed a 1:2:1 segregation ratio of AA:AB:BB allele, which suggested the possibility of deriving loci from candidate subgenome-specific sequences. In addition, 2575 loci (2414 SNPs and 161 SSR loci) showed a 3:1 segregation of AB:BB allele, indicating they were derived from homoeologous genomic sequences. Comparative analysis of the homoeologous linkage groups revealed differences in genome structure among the subgenomes.ConclusionsOur results suggest that candidate subgenome-specific loci are randomly located across the genomes, and that there are small- to large-scale structural variations among the subgenomes. The mapped SNPs and SSR loci on the linkage map are expected to be seed points for the construction of pseudomolecules in the octoploid strawberry.

MiRNA-556-3p promotes human bladder cancer proliferation, migration and invasion by negatively regulating DAB2IP expression.

MicroRNAs (miRNAs) play critical roles in tumorigenesis and metastasis by negatively regulating gene expression through complementary binding to the 3'-untranslated region of target mRNAs. The role of miRNAs in expression of the tumor suppressor DAB2IP in bladder cancer (BC) remains unknown. The aim of the present study was to identify miRNAs targeting DAB2IP and determine their expression and function in BC. We predicted candidate miRNAs targeting DAB2IP using TargetScan software. Dual-luciferase reporter assays confirmed that miRNA-556-3p directly regulated DAB2IP expression. Quantitative RT-PCR and RNase protection assays showed that endogenous miRNA-556-3p expression was significantly upregulated in clinical samples of BC patients and BC cell lines and western blot analysis indicated that DAB2IP expression in BC tissues and BC cell lines was concurrently downregulated. Gain or loss of function studies showed that upregulation of miRNA-556-3p promoted proliferation, invasion, migration and colony formation of BC cells, whereas downregulation resulted in opposite effects. Importantly, restoration of DAB2IP expression rescued the effects induced by miRNA-556-3p. Overexpression of miRNA-556-3p in BC cells not only decreased DAB2IP expression, but also markedly increased Ras GTPase activity and ERK1/2 phosphorylation level. These findings suggest that DAB2IP is a direct target of miRNA-556-3p, and endogenous miRNA-556-3p expression shows inverse correlation with simultaneous DAB2IP expression in BC tissues and cells. miRNA-556-3p functions as a tumor promoter in tumorigenesis and metastasis of BC by targeting DAB2IP. Moreover, miRNA-556-3p-mediated DAB2IP suppression plays an oncogenic role by partial activation of the Ras-ERK pathway.

Identification and characterization of two novel PTCH1 splice variants

Patched-1 (PTCH1), one of the key molecules involved in the Hedgehog (HH) signaling pathway, acts as the receptor of the HH ligand. PTCH1 also inhibits the positive signal transducer Smoothened (SMO). Several PTCH1 splice variants have been identified and confirmed to play critical roles in HH pathway regulation. In the present study, two novel alternatively spliced variants of PTCH1 transcripts, designated PTCH1-Δ10 and PTCH1-Δ15, were found in humans, mice and zebrafish using RT-PCR, direct sequencing and ribonuclease protection assays. PTCH1-Δ10 lacks exon 10, which encodes part of the sterol-sensing domain (SSD), while PTCH1-Δ15 lacks 166 bp of exon 15, which causes a frame shift that generates a premature stop codon leading to a truncated PTCH1 protein. Different truncated PTCH1 proteins localized in the cytoplasm were capable of internalizing the N-terminal fragment of Sonic hedgehog (SHH-N), which was visualized using immunofluorescence microscopy. Exon skipping dramatically influenced the steady states of the proteins, with the levels of PTCH1-1B and PTCH1-Δ10 being significantly higher than those of PTCH1-Δ15, as detected using western blot. These results imply that the pronounced inhibitory signaling properties of PTCH1-1B and PTCH1-Δ10 may be partially due to high protein expression in addition to intrinsic functional differences.All isoforms examined worked as functional receptors of SHH. However, the isoforms PTCH1-1B and PTCH1-Δ10 inhibited SMO and the pathway transcription factor glioma 1 (GLI1) to a greater extent than did PTCH1-Δ15. In addition, PTCH1-1B and PTCH1-Δ10 (but not PTCH1-Δ15) can be negative regulators of the HH pathway. These results indicate that the SSD domain and the C-terminal region are essential for maximal repressor function of PTCH1. Additionally, SMO inhibition by PTCH1 occurred through a nonstoichiometric, catalytic mechanism, indicating that this inhibition was less dependent on the dose of the PTCH1 protein. Finally, all these isoforms have been revealed to inhibit GLI1 activation by either the classical HH signaling pathway or a new pathway not reliant on both SMO and apoptosis. Thus, our study clearly demonstrated the unique involvement of the two novel PTCH1 splice variants in HH signal transduction.

Validation of a Multiplexed Gene Signature Assay for Diagnosis of Canine Cancers from Formalin‐Fixed Paraffin‐Embedded Tissues

BackgroundUse of molecular‐based diagnostics for companion animals is impeded by availability of technology platforms, tissue acquisition requirements, and species‐specific reagents.Hypothesis/ObjectivesTo validate a quantitative nuclease protection assay (qNPA) to simultaneously measure RNA expression of multiple genes in archived formalin‐fixed paraffin‐embedded (FFPE) tumors from dogs.AnimalsAll tumor biopsy samples were collected retrospectively from surgical biopsies and in the care of veterinarians.MethodsRetrospective case series. A qNPA 96‐well ArrayPlate was built using 30 canine‐specific genes, 5 housekeeping genes, positive and negative controls with qualified gene‐specific oligonucleotides. Pearson's correlation, coefficient of variation (CV), and multivariate analysis were used to determine analytical performance using 40 FFPE dog tumors. Once validated, 70 FFPE dog tumors were analyzed for differences in gene expression using hierarchical clustering and analysis of variance of log transformed data. Immunohistochemistry (IHC) was performed to correlate gene expression and protein expression in a subset of tumors.ResultsThe assay was linear with decreasing sample input (R 2 = 0.978), reproducible within and between 96‐well plates (r = 0.988 and 0.95, respectively) and between different laboratories (CV = 0.96). Hierarchical cluster analysis showed grouping of tumors by histogenesis and oncogenes. Significant differences were found between BCl2, E2F transcription factor 1, MDM2, COX‐2, MET proto‐oncogene receptor kinase, and other biologically relevant gene expression in tumor subtypes. Immunohistochemistry confirmed protein expression.Conclusions and Clinical ImplicationsBecause this technology works reliably on FFPE specimens, it can help expedite the broad introduction of multiplexed genomic information for improved diagnostics and discovery of new targets for therapies in veterinary oncology.

A high-density SNP genetic map consisting of a complete set of homologous groups in autohexaploid sweetpotato (Ipomoea batatas)

Sweetpotato (Ipomoea batatas) is an autohexaploid species with 90 chromosomes (2n = 6x = 90) and a basic chromosome number of 15, and is therefore regarded as one of the most challenging species for high-density genetic map construction. Here, we used single nucleotide polymorphisms (SNPs) identified by double-digest restriction site-associated DNA sequencing based on next-generation sequencing technology to construct a map for sweetpotato. We then aligned the sequence reads onto the reference genome sequence of I. trifida, a likely diploid ancestor of sweetpotato, to detect SNPs. In addition, to simplify analysis of the complex genetic mode of autohexaploidy, we used an S1 mapping population derived from self-pollination of a single parent. As a result, 28,087 double-simplex SNPs showing a Mendelian segregation ratio in the S1 progeny could be mapped onto 96 linkage groups (LGs), covering a total distance of 33,020.4 cM. Based on the positions of the SNPs on the I. trifida genome, the LGs were classified into 15 groups, each with roughly six LGs and six small extra groups. The molecular genetic techniques used in this study are applicable to high-density mapping of other polyploid plant species, including important crops.

Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction.

Coding region determinant-binding protein (CRD-BP) binds to the 3’-UTR of microphthalmia-associated transcription factor (MITF) mRNA to prevent its targeted degradation by miR-340. Here, we aim to further understand the molecular interaction between CRD-BP and MITF RNA. Using point mutation in the GXXG motif of each KH domains, we showed that all four KH domains of CRD-BP are important for their physical association with MITF RNA. We mapped the CRD-BP-binding site in the 3’-UTR of MITF RNA from nts 1330–1740 and showed that the 49-nt fragment 1621–1669 is the minimal size MITF RNA for binding. Upon deletion of nts 1621–1669 within the nts1550-1740 of MITF RNA, there was a 3-fold increase in dissociation constant Kd, which further confirms the critical role sequences within nts 1621–1669 in binding to CRD-BP. Amongst the eight antisense oligonucleotides designed against MITF RNA 1550–1740, we found MHO-1 and MHO-7 as potent inhibitors of the CRD-BP-MITF RNA interaction. Using RNase protection and fluorescence polarization assays, we showed that both MHO-1 and MHO-7 have affinity for the MITF RNA, suggesting that both antisense oligonucleotides inhibited CRD-BP-MITF RNA interaction by directly binding to MITF RNA. The new molecular insights provided in this study have important implications for understanding the oncogenic function of CRD-BP and development of specific inhibitors against CRD-BP-MITF RNA interaction.

ATPase activity tightly regulates RecA nucleofilaments to promote homologous recombination

Homologous recombination (HR), catalyzed in an evolutionarily conserved manner by active RecA/Rad51 nucleofilaments, maintains genomic integrity and promotes biological evolution and diversity. The structures of RecA/Rad51 nucleofilaments provide information critical for the entire HR process. By exploiting a unique capillary electrophoresis-laser-induced fluorescence polarization assay, we have discovered an active form of RecA nucleofilament, stimulated by ATP hydrolysis, that contains mainly unbound nucleotide sites. This finding was confirmed by a nuclease protection assay and electron microscopy (EM) imaging. We further found that these RecA-unsaturated filaments promote strand exchange in vitro and HR in vivo. RecA mutants (P67D and P67E), which only form RecA-unsaturated nucleofilaments, were able to mediate HR in vitro and in vivo, but mutants favoring the formation of the saturated nucleofilaments failed to support HR. We thus present a new model for RecA-mediated HR in which RecA utilizes its intrinsic DNA binding-dependent ATPase activity to remodel the nucleofilaments to a less saturated form and thereby promote HR.

Isolation of Polysomal RNA for Analyzing Stress-Responsive Genes Regulated at the Translational Level in Plants.

Alteration of gene expression is an essential mechanism, which allows plants to respond and adapt to adverse environmental conditions. Transcriptome and proteome analyses in plants exposed to abiotic stresses revealed that protein levels are not correlated with the changes in corresponding mRNAs, indicating regulation at translational level is another major regulator for gene expression. Analysis of translatome, which refers to all mRNAs associated with ribosomes, thus has the potential to bridge the gap between transcriptome and proteome. Polysomal RNA profiling and recently developed ribosome profiling (Ribo-seq) are two main methods for translatome analysis at global level. Here, we describe the classical procedure for polysomal RNA isolation by sucrose gradient ultracentrifugation followed by highthroughput RNA-seq to identify genes regulated at translational level. Polysomal RNA can be further used for a variety of downstream applications including Northern blot analysis, qRT-PCR, RNase protection assay, and microarray-based gene expression profiling.

Evaluation and selection of a non-PCR based technology for improved gene expression profiling from clinical formalin-fixed, paraffin-embedded samples.

Formalin-fixed, paraffin-embedded (FFPE) clinical tissue samples have the potential to provide valuable gene-expression data for the development of cancer biomarkers. However, FFPE RNA is extensively fragmented, presenting a significant challenge for reliably detecting gene expression using traditional qPCR methods. We evaluated three novel methodologies along with the traditional qPCR method for their ability to detect Notch pathway gene expression in colorectal cancer FFPE tissue RNAs. We found that quantitative nuclease protection assay-detected gene expression in high-quality RNAs as sensitively as qPCR, and consistently detected mRNAs in highly fragmented FFPE tissue RNAs. Quantitative nuclease protection assay represents an improved methodology for detecting gene expression in FFPE tissue and has the potential to advance the development of cancer biomarkers.

An efficient and rapid method to detect and verify natural antisense transcripts of animal genes

High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lncRNA (long non-coding RNA), can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix (Xist antisense RNA), chicken LXN (latexin) and GFM1 (G elongation factor, mitochondrial 1), were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.

From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging.

In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community.

Association of tumor and plasma microRNA expression with tumor monosomy-3 in patients with uveal melanoma.

BackgroundEpigenetic events mediated by methylation and histone modifications have been associated with the development of metastasis in patients with uveal melanoma. The role of epigenetic events mediated by microRNA (miR) is less clear. Tumor and plasma miR expression was examined in patients with primary uveal melanoma with tumor monosomy-3, a predictor of metastasis.ResultsmiR profiling of tumors by microarray found six miRs over-expressed and 19 under-expressed in 33 tumors with monosomy-3 compared to 22 without. None of the miRs differentially expressed in tumors with and without monosomy-3 was differentially expressed in tumors with and without tumor infiltrating lymphocytes. Tumors manifesting monosomy-3 were also characterized by higher levels of TARBP2 and DDX17 and by lower levels of XPO5 and HIWI, miR biogenesis factors. miR profiling of plasma by a quantitative nuclease protection assay found elevated levels of 11 miRs and reduction in four in patients with tumor monosomy-3. Only three miRs differentially expressed in the tumor arrays were detectable in plasma. miRs implicated in uveal melanoma development were not differentially expressed. Elevated plasma levels in patients with tumor monosomy-3 of miR-92b, identified in the tumor array, and of miR-199-5p and miR-223, identified in the plasma array, were confirmed by quantitative real-time polymerase chain reaction. Levels were also higher in patients compared to normal controls.ConclusionsThese results support a role for epigenetic mechanisms in the development of metastasis in patients with uveal melanoma and the analysis of miRs as biomarkers of metastatic risk. They also suggest that potentially useful blood miRs may be derived from the host response as well as the tumor.

Abstract 2237: Gene expression measurement of immuno-oncology targets in a single FFPE section using a novel targeted sequencing assay

Abstract Background and Purpose: The field of immuno-oncology (IO) covers a broad set of research disciplines and presents a highly diverse set of experimental requirements. The challenges include sample types of varying quality and quantity of material (including both small fixed samples and blood products) as well as an expanding multiplicity of targets to assay for immunological response. The HTG EdgeSeq system combines HTG's proprietary quantitative nuclease protection assay chemistry with a Next Generation Sequencing (NGS) platform to enable semiquantitative analysis of hundreds of targeted genes in a single assay. The novel HTG EdgeSeq Immuno-Oncology Assay measures 549 IO-related genes and can be used with most clinically relevant samples. Methods: An internal verification study was performed to evaluate a five-point, two-fold titration series (6 concentrations) in a variety of sample types with as few as a 250 cells, 32 μl of PAXgene, or 1.56 mm2 of a 5 μm FFPE section. FFPE sample types included: melanoma; DLBCL; and tissue from lung, breast, colon and renal carcinomas. Additionally, examples of the biological relevance of this data are provided by examination of expression from several pairs of samples, each profiled using a single 5 μm FFPE section. Results: Equivalent expression across the dynamic range was obtained within each of the sample types (Pearson correlations ranging between 0.84 to 0.98). For DLBCL, while most canonical markers of immune cells showed similar patterns of expression, different patterns of expression of T cell and NK cell genes were obtained, likely indicating a different composition of the immune cell infiltrates within these tumors. Specific to the two different melanoma tumors the lymphocyte infiltrates appear to be similar in these tumors. Interestingly, a markedly different immune response appears to be occurring in the melanoma tumors. One tumor appears to be mounting a significant type I interferon response, which is not as apparent in the second tumor. Differential expression of other well-known metastasis-associated genes are also observed between the two tumors. These observations suggest that patterns of differential expression could be used to assist in directing patients to targeted therapies. Conclusion: The HTG EdgeSeq Immuno-Oncology Assay provides a valuable tool for researchers exploring the immune response to tumors across a wide variety of tissues. Combining highly reproducible results with very small sample input amounts allows the assay to be utilized for the very small and precious samples available to researchers in this field. Citation Format: Monica M. Reinholz, Debrah Thompson, James Cooley, Xiao-Bo Chen, Iris Howlett, John Luecke, Patrick Roche, Bonnie LaFleur. Gene expression measurement of immuno-oncology targets in a single FFPE section using a novel targeted sequencing assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2237.

Genetic Approaches to Study Plant Responses to Environmental Stresses: An Overview.

The assessment of gene expression levels is an important step toward elucidating gene functions temporally and spatially. Decades ago, typical studies were focusing on a few genes individually, whereas now researchers are able to examine whole genomes at once. The upgrade of throughput levels aided the introduction of systems biology approaches whereby cell functional networks can be scrutinized in their entireties to unravel potential functional interacting components. The birth of systems biology goes hand-in-hand with huge technological advancements and enables a fairly rapid detection of all transcripts in studied biological samples. Even so, earlier technologies that were restricted to probing single genes or a subset of genes still have their place in research laboratories. The objective here is to highlight key approaches used in gene expression analysis in plant responses to environmental stresses, or, more generally, any other condition of interest. Northern blots, RNase protection assays, and qPCR are described for their targeted detection of one or a few transcripts at a once. Differential display and serial analysis of gene expression represent non-targeted methods to evaluate expression changes of a significant number of gene transcripts. Finally, microarrays and RNA-seq (next-generation sequencing) contribute to the ultimate goal of identifying and quantifying all transcripts in a cell under conditions or stages of study. Recent examples of applications as well as principles, advantages, and drawbacks of each method are contrasted. We also suggest replacing the term “Next-Generation Sequencing (NGS)” with another less confusing synonym such as “RNA-seq”, “high throughput sequencing”, or “massively parallel sequencing” to avoid confusion with any future sequencing technologies.

Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis

During the entry process, the human papillomavirus (HPV) capsid is trafficked to the trans-Golgi network (TGN), whereupon it enters the nucleus during mitosis. We previously demonstrated that the minor capsid protein L2 assumes a transmembranous conformation in the TGN. Here we provide evidence that the incoming viral genome dissociates from the TGN and associates with microtubules after the onset of mitosis. Deposition onto mitotic chromosomes is L2-mediated. Using differential staining of an incoming viral genome by small molecular dyes in selectively permeabilized cells, nuclease protection, and flotation assays, we found that HPV resides in a membrane-bound vesicle until mitosis is completed and the nuclear envelope has reformed. As a result, expression of the incoming viral genome is delayed. Taken together, these data provide evidence that HPV has evolved a unique strategy for delivering the viral genome to the nucleus of dividing cells. Furthermore, it is unlikely that nuclear vesicles are unique to HPV, and thus we may have uncovered a hitherto unrecognized cellular pathway that may be of interest for future cell biological studies.