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Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples
Low RNA yield and quality limit use of formalin-fixed paraffin-embedded (FFPE) tissue samples for genomic analyses. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and target gene responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n=6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 hours followed by ethanol (18F); and 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. The latter group received no additional treatment (3F) or the following demodification protocols: short heated incubation with TAE buffer; overnight heated incubation with an organocatalyst using two different isolation kits; or overnight heated incubation without organocatalyst. TruSeq Stranded Total RNA libraries with Ribo-Zero were built and sequenced using the Illumina HiSeq platform. Extended incubation with or without organocatalyst increased RNA yield >3-fold and enhanced quality compared to 3F, as indicated by higher RNA integrity number (>1.5-fold) and fragment analysis values (>3.0-fold). Post-sequencing metrics showed reduced bias in gene coverage and deletion rates for all extended incubation groups. Following PB-induced differential gene expression analysis, all demodification groups showed increased overlap with FR in genes (73-83%) and pathways (91-94%) compared to 3F overlap with FR (60% and 63%, respectively). These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples. This dataset is associated with the following publication: Wehmas, L., C. Wood, R. Gagne, A. Williams, C. Yauk, M. Gosink, D. Dalmas, R. Hao, R. O'Lone, and S. Hester. Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 162(2): 535-547, (2018).
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Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded Samples.
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se of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-seq offers a novel way to address this problem. In this study we evaluated transcriptomic dose responses using RNA-seq in paired FFPE and frozen (FROZ) samples from two archival studies in mice, one recent (<2 years old) and the other older (>20 years old). Experimental treatments included di(2-ethylhexyl)phthalate (DEHP) and dichloroacetic acid (DCA) for the <2 and >20 year-old studies, respectively. Total RNA was ribodepleted and sequenced using the Illumina HiSeq platform. In the recent study, FFPE samples showed high concordance in total reads (98% vs FROZ), fold-change values of differentially expressed genes (DEGs) (R2 = 0.99), highly enriched target pathways (90% overlap with FROZ), and benchmark dose estimates for preselected target genes (-2% overall vs FROZ). In contrast, RNA-seq data from older FFPE samples had lower total reads (70% vs FROZ) and poor concordance in global DEGs and pathways. Despite a 99% loss of counts, dose responses were still evident for target genes in FFPE samples and positively correlated with paired FROZ samples. These findings highlight potential variability in the quality of RNA-seq data from FFPE samples. More recent FFPE samples were highly similar to FROZ samples in sequencing quality metrics, DEG profiles, and dose-response parameters, while further methods development is needed for older or lower-quality FFPE samples. This work should help broaden the use of archival resources in both chemical safety and translational science. This dataset is associated with the following publication: Hester, S., V. Bhat, B. Chorley, G. Carswell, W. Jones, L. Wehmas, and C. Wood. Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples.. TOXICOLOGICAL SCIENCES. Society of Toxicology, 154(2): 202-213, (2016).
Datasets associated with journal article 'Combining phenotypic profiling and targeted RNA-Seq reveals linkages between transcriptional perturbations and chemical effects on cell morphology: Retinoic acid as an example' by Nyffeler, J, et.al.
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We evaluated the Templated Oligo with Sequencing Readout (TempO-Seq) assay for High-throughput transcriptomic screening of a small set of environmental chemicals. This assay yields sequencing reads of exactly 50 base pairs that can be rapidly aligned to generate gene counts, and is compatible with cell lysates prepared in multiwell format. The version of the TempO-Seq assay we evaluated provides nearly whole transcriptome coverage (>20,000 genes). This study encompasses 2 replicates of a 384-well plate design. The majority of wells contain U-2 OS cells exposed to 11 test chemicals at 7 different concentrations (two replicate per test chemical, concentration, and plate), and additional reference samples and controls. Controls include DMSO vehicle treatments (22 per plate). Reference samples include bulk lysate MCF7 samples (2 DMSO treated and 2 TSA treated samples per plate), reference chemical treatments (3 chemicals at single conc each, per plate), and vendor-provided reference RNA mixtures (UHRR and HBRR). This dataset is associated with the following publication: Nyffeler, J., C. Willis, D. Harris, L. Taylor, R. Judson, L. Everett, and J. Harrill. Combining phenotypic profiles and targeted RNA-Seq reveals linkages between transcriptional perturbations and chemical effects on cell morphology: retinoic acid as an example.. TOXICOLOGY AND APPLIED PHARMACOLOGY. Academic Press Incorporated, Orlando, FL, USA, 444: 116032, (2022).
Noncoding RNA gene detection using comparative sequence analysis
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Background Noncoding RNA genes produce transcripts that exert their function without ever producing proteins. Noncoding RNA gene sequences do not have strong statistical signals, unlike protein coding genes. A reliable general purpose computational genefinder for noncoding RNA genes has been elusive. Results We describe a comparative sequence analysis algorithm for detecting novel structural RNA genes. The key idea is to test the pattern of substitutions observed in a pairwise alignment of two homologous sequences. A conserved coding region tends to show a pattern of synonymous substitutions, whereas a conserved structural RNA tends to show a pattern of compensatory mutations consistent with some base-paired secondary structure. We formalize this intuition using three probabilistic "pair-grammars": a pair stochastic context free grammar modeling alignments constrained by structural RNA evolution, a pair hidden Markov model modeling alignments constrained by coding sequence evolution, and a pair hidden Markov model modeling a null hypothesis of position-independent evolution. Given an input pairwise sequence alignment (e.g. from a BLASTN comparison of two related genomes) we classify the alignment into the coding, RNA, or null class according to the posterior probability of each class. Conclusions We have implemented this approach as a program, QRNA, which we consider to be a prototype structural noncoding RNA genefinder. Tests suggest that this approach detects noncoding RNA genes with a fair degree of reliability.
Retrovirus-delivered siRNA
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The ability of transfected synthetic small interfering (si) RNAs to suppress the expression of specific transcripts has proved a useful technique to probe gene function in mammalian cells. However, high production costs limit this technology's utility for many laboratories and experimental situations. Recently, several DNA-based plasmid vectors have been developed that direct transcription of small hairpin RNAs, which are processed into functional siRNAs by cellular enzymes. Although these vectors provide certain advantages over chemically synthesized siRNAs, numerous disadvantages remain including merely transient siRNA expression and low and variable transfection efficiency.
['"Molecular Transducers of Human Skeletal Muscle Remodeling under Different Loading States"']
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['"The annotation of the Affymetrix HTA 2.0 array was updated to optimise the detection of RNA in human skeletal muscle biopsy samples by removing invalid and low signal-high-variance probes (as for CDF GPL24047). The probes were then summarized into groups (probe-sets) reflecting either an ensembl full transcript identifier (FL-ENST, GPL24047) or just the probes targeting the 3\' UTR or the 5\' UTR of that particular ENST. Therefore, 3 different CDF were used to process the HTA 2.0 arrays in this study. Note that each CEL file was GC adjusted using APT while our custom CDF pipeline removes any probe that has >80% or <20% GC content (~50,000). The analysis was carried out only on the pairs of probe-sets i.e. FL-ENST vs 3\'UTR or FL-ENST vs 5\'UTR or 3\'UTR vs 5\'UTR. Dynamic muscle loading alters tissue phenotype reflecting altered metabolic and functional demands. In humans, heterogeneous adaptation to loading complicates identification of the underpinning molecular regulators. We present a within-person analysis strategy that reduced heterogeneity for changes in muscle mass by ~40% and employed a genome-wide transcriptome method that modeled each mRNA from coding exons and 3’/5’ untranslated (UTR) regions. Our strategy detected ~3-4 times more regulated genes than similarly sized studies, including substantial UTR-selective regulation that other methods would not detect. We discovered a core of 141 genes correlated to muscle growth validated from newly analyzed independent samples (n=100). Further validating these identified genes, via RNAi in primary muscle cells, we demonstrate that members of the core genes were regulators of protein synthesis e.g. Molecular Transducers of Physical Activity in Humans MoTrPAC. Employing proteome-constrained networks and pathway analysis revealed notable relationships with the molecular characteristics of human muscle aging and insulin sensitivity, as well as potential drug-therapies."']
Quantitative assessment of the use of modified nucleoside triphosphates in expression profiling: differential effects on signal intensities and impacts on expression ratios
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Background The power of DNA microarrays derives from their ability to monitor the expression levels of many genes in parallel. One of the limitations of such powerful analytical tools is the inability to detect certain transcripts in the target sample because of artifacts caused by background noise or poor hybridization kinetics. The use of base-modified analogs of nucleoside triphosphates has been shown to increase complementary duplex stability in other applications, and here we attempted to enhance microarray hybridization signal across a wide range of sequences and expression levels by incorporating these nucleotides into labeled cRNA targets. Results RNA samples containing 2-aminoadenosine showed increases in signal intensity for a majority of the sequences. These results were similar, and additive, to those seen with an increase in the hybridization time. In contrast, 5-methyluridine and 5-methylcytidine decreased signal intensities. Hybridization specificity, as assessed by mismatch controls, was dependent on both target sequence and extent of substitution with the modified nucleotide. Concurrent incorporation of modified and unmodified ATP in a 1:1 ratio resulted in significantly greater numbers of above-threshold ratio calls across tissues, while preserving ratio integrity and reproducibility. Conclusions Incorporation of 2-aminoadenosine triphosphate into cRNA targets is a promising method for increasing signal detection in microarrays. Furthermore, this approach can be optimized to minimize impact on yield of amplified material and to increase the number of expression changes that can be detected.
Rodent Research-1 (RR1) NASA Validation Flight: Mouse liver transcriptomic proteomic and epigenomic data
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RR-1 is a validation flight to evaluate the hardware operational and science capabilities of the Rodent Research Project on the ISS. RNA DNA and protein were purified from liver tissues from RR-1 mice (female C57Bl6/J 16wk old at time of launch) including eight from the Flight group and eight from the Ground Control group. From each group two liver samples were collected and frozen immediately after euthanasia (Flight mice dissected on-orbit after total 37 days after launch Samples FLT-M21 M22 and corresponding Ground Control samples GC-M31,M32). An additional six samples from each group were collected from frozen carcasses dissected post-flight (Samples FLT-M25,M26,M27,M28,M29,M30 and corresponding Ground Control samples GC-M35,M36,M37,M38,M39,M40). RNA-Seq whole genome and RNA BS-Seq (bisulfite sequencing) and proteomic expression profiling were performed.
Rodent Research-1 (RR1) National Lab Validation Flight: Mouse liver transcriptomic proteomic and epigenomic data
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The Rodent Reasearch-1 National Lab (RR-1 CASIS) experiment was performed to study the effect of microgravity on muscle wasting. RNA DNA and protein were purified from RR-1 CASIS (the Center for the Advancement of Science in Space) liver samples. Groups included: Flight (FLT) dissected on-orbit (21 or 22 days after launch); age-matched Ground Controls (GC); and Basal Controls (BC euthanized at time of launch). RNA-Seq whole genome BS-Seq (bisulfite sequencing) and proteomic expression profiling were performed.
Rodent Research-1 (RR1) NASA Validation Flight: Mouse soleus muscle transcriptomic and epigenomic data
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NASA s Rodent Research (RR) project is playing a critical role in advancing biomedical research on the physiological effects of space environments. Due to the limited resources for conducting biological experiments aboard the International Space Station (ISS) it is imperative to use crew time efficiently while maximizing high-quality science return. NASA s GeneLab project has as its primary objectives to 1) further increase the value of these experiments using a multi-omics systems biology-based approach and 2) disseminate these data without restrictions to the scientific community. The current investigation assessed viability of RNA DNA and protein extracted from archived RR-1 tissue samples for epigenomic transcriptomic and proteomic assays. During the first RR spaceflight experiment a variety of tissue types were harvested from subjects snap-frozen or RNAlater-preserved and then stored at least a year at -80C after return to Earth. They were then prioritized for this investigation based on likelihood of significant scientific value for spaceflight research. All tissues were made available to GeneLab through the bio-specimen sharing program managed by the Ames Life Science Data Archive and included mouse adrenal glands quadriceps gastrocnemius tibialis anterior extensor digitorum longus soleus eye and kidney. We report here protocols for and results of these tissue extractions and thus the feasibility and value of these kinds of omics analyses. In addition to providing additional opportunities for investigation of spaceflight effects on the mouse transcriptome and proteome in new kinds of tissues our results may also be of value to program managers for the prioritization of ISS crew time for rodent research activities.
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