Normalization of Full-Length-Enriched cDNA

Ea, Bogdanova; Ev, Barsova; Shagina, Irina; A, Scheglov; Anisimova,; Ll, Vagner; Sa, Lukyanov; Da, Shagin

doi:10.1007/978-1-61779-065-2_6

Cited by 27 publications

(15 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Whether finding the causative mutations or simply cataloguing the wealth of expressed genes is a priority, normalizing the RNA extract is beneficial [46,47]. In agreement with previous studies, we found that normalized samples produced the highest number of contigs but also singleton reads, which are equivalent to rare transcripts that might harbor valuable information otherwise not accessible [16,48].…”

Section: Discussionsupporting

confidence: 87%

Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

et al. 2012

View full text Add to dashboard Cite

Background Since the emergence of next generation sequencing platforms, unprecedented opportunities have arisen in the study of natural vertebrate populations. In particular, insights into the genetic and epigenetic mechanisms of adaptation can be revealed through study of the expression profiles of genes. However, as a pre-requisite to expression profiling, care must be taken in RNA preparation as factors like DNA contamination, RNA integrity or transcript abundance can affect downstream applications. Here, we evaluated five commonly used RNA extraction methods using whole blood sampled under varying conditions from 20 wild carnivores. Results Despite the use of minute starting volumes, all methods produced quantifiable RNA extracts (1.4 – 18.4 μg) with varying integrity (RIN 4.6 - 7.7), the latter being significantly affected by the storage and extraction method used. We observed a significant overall effect of the extraction method on DNA contamination. One particular extraction method, the LeukoLOCK™ filter system, yielded high RNA integrity along with low DNA contamination and efficient depletion of hemoglobin transcripts highly abundant in whole blood. In a proof of concept sequencing experiment, we found globin RNA transcripts to occupy up to ¼ of all sequencing reads if libraries were not depleted of hemoglobin prior to sequencing. Conclusion By carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species.

show abstract

Section: Discussionsupporting

confidence: 87%

Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

et al. 2012

View full text Add to dashboard Cite

show abstract

“…Such transcripts are beyond reliable detection using the types of libraries and methods we report here. Additional methods such as subcellular fractionation, cell type isolation, and library normalization or targeted enrichment will be required to reach the bottom of the transcriptome (Kapranov et al 2007;Bogdanova et al 2008).…”

Section: Spike-in Controls For Rna-seqmentioning

confidence: 99%

Synthetic spike-in standards for RNA-seq experiments

Jiang¹,

Schlesinger²,

Davis³

et al. 2011

Genome Res.

610

548

View full text Add to dashboard Cite

High-throughput sequencing of cDNA (RNA-seq) is a widely deployed transcriptome profiling and annotation technique, but questions about the performance of different protocols and platforms remain. We used a newly developed pool of 96 synthetic RNAs with various lengths, and GC content covering a 2 20 concentration range as spike-in controls to measure sensitivity, accuracy, and biases in RNA-seq experiments as well as to derive standard curves for quantifying the abundance of transcripts. We observed linearity between read density and RNA input over the entire detection range and excellent agreement between replicates, but we observed significantly larger imprecision than expected under pure Poisson sampling errors. We use the control RNAs to directly measure reproducible protocol-dependent biases due to GC content and transcript length as well as stereotypic heterogeneity in coverage across transcripts correlated with position relative to RNA termini and priming sequence bias. These effects lead to biased quantification for short transcripts and individual exons, which is a serious problem for measurements of isoform abundances, but that can partially be corrected using appropriate models of bias. By using the control RNAs, we derive limits for the discovery and detection of rare transcripts in RNA-seq experiments. By using data collected as part of the model organism and human Encyclopedia of DNA Elements projects (ENCODE and modENCODE), we demonstrate that external RNA controls are a useful resource for evaluating sensitivity and accuracy of RNA-seq experiments for transcriptome discovery and quantification. These quality metrics facilitate comparable analysis across different samples, protocols, and platforms.[Supplemental material is available for this article.]High-throughput sequencing applications are revolutionizing genome-wide analysis (Mardis 2008;Mortazavi et al. 2008;Celniker et al. 2009;Morozova et al. 2009;Gerstein et al. 2010;Metzker 2010;Roy et al. 2010). RNA-seq offers single-nucleotide resolution, strand specificity, and short-range connectivity through pairedend sequencing. Because of these strengths, there has been great interest in using RNA-seq to distinguish isoforms, calculate expression levels for transcripts, and uncover low abundance RNAs (He et al. 2008;Mortazavi et al. 2008;Nagalakshmi et al. 2008;Sultan et al. 2008;Wang et al. 2008Wang et al. , 2010Passalacqua et al. 2009;Gerstein et al. 2010;Roy et al. 2010;Trapnell et al. 2010;Berezikov et al. 2011;Graveley et al. 2011).While there are clear advantages to RNA-seq, it is less clear how well the procedure performs, as several studies have reported conflicting RNA-seq accuracy results. RNA-seq-determined concentrations of six in vitro synthetic transcripts show good linearity (Mortazavi et al. 2008), and in a study using quantitative PCR as the benchmark, RNA-seq showed better performance for genes with high expression, while two-channel microarrays were more sensitive in identifying differential expression between genes with low ex...

show abstract

“…Email: endeldw @ uc.edu DNA has also been applied for many techniques using short RNA sequences [16,[18][19][20][21][22][23][24][25][26][27][28][29], but applications for long RNA targets have remained elusive.…”

Section: * Corresponding Authormentioning

confidence: 99%

Rational Design of Duplex Specific Nuclease for One-Step Isothermal Viral RNA Detection

2017

J App Biol Biotech

View full text Add to dashboard Cite

RNA viruses are a potent human adversary, evidenced by several global pandemics including the Ebolavirus in West Africa, the emerging Zika virus, and outbreaks of new Influenza strains and Norwalk virus in the food supply and cruise ships. Despite the virulence of these pathogens, there remains a significant limitation for detecting these viruses in a fast, accurate and cost effective manner. To meet this need we present a modified form of the duplex specific nuclease enzyme from the Paralithodes camtschaticus crab capable of generating an RNA-based signal amplification in a fraction of the time required for standard RT-qPCR. The applicability of this enzyme is demonstrated in an assay for Norwalk virus detection with a lower limit of ~100 viral copies per liter of environmental water.

show abstract

Normalization of Full-Length-Enriched cDNA

Cited by 27 publications

References 20 publications

Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

Synthetic spike-in standards for RNA-seq experiments

Rational Design of Duplex Specific Nuclease for One-Step Isothermal Viral RNA Detection

Contact Info

Product

Resources

About