Multiplexed hydrogel microparticle suspension arrays for facile ribosomal RNA integrity assays

Abstract Background: Both RNA-Seq and sample freeze-thaw are ubiquitous. However, knowledge about the impact of freeze-thaw on downstream analyses is limited. The lack of common quality metrics that are sufficiently sensitive to freeze-thaw and RNA degradation, e.g. the RNA Integrity Score, makes such assessments challenging.Results: Here we quantify the impact of repeated freeze-thaw cycles on the reliability of RNA-Seq by examining poly(A)-enriched and ribosomal RNA depleted RNA-seq from frozen leukocytes drawn from a toddler Autism cohort. To do so, we estimate the relative noise, or percentage of random counts, separating technical replicates. Using this approach we measured noise associated with RIN and freeze-thaw cycles. As expected, RIN does not fully capture sample degradation due to freeze-thaw. We further examined differential expression results and found that three freeze-thaws should extinguish the differential expression reproducibility of similar experiments. Freeze-thaw also resulted in a 3’ shift in the read coverage distribution along the gene body of poly(A)-enriched samples compared to ribosomal RNA depleted samples, suggesting that library preparation may exacerbate freeze-thaw-induced sample degradation.Conclusion: The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility.

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Changes in ribosomal RNA integrity in leek (Allium porrum L.) seeds during osmopriming and drying-back treatments

Seed Science Research ◽

10.1017/s0960258500000611 ◽

1991 ◽

Vol 1 (1) ◽

pp. 37-44 ◽

Cited By ~ 5

Author(s):

P. A. Davison ◽

R. M. Taylor ◽

C. M. Bray

Keyword(s):

Protein Synthesis ◽

Polyethylene Glycol ◽

Ribosomal Rna ◽

Rna Degradation ◽

Allium Porrum ◽

Rna Integrity ◽

Embryo Tissue ◽

Synthetic Capacity ◽

Ribosomal Rrna ◽

Tolerant State

AbstractLoss of vigour in leek (Allium porrum L.) seed lots is accompanied by the appearance of damage to ribosomal RNA in quiescent embryo tissue. Polyethylene glycol osmopriming treatments of such low-vigour seed permit replacement of this damaged ribosomal rRNA over a 7-day priming period. Low-vigour leek seeds germinated for 6 days without a prior osmopriming treatment still exhibit evidence of damaged ribosomal RNA in embryo tissue. Osmoprimed leek seeds dried back whilst still in the desiccation-tolerant state retain most of the benefits conferred by priming treatments. Osmoprimed leek seeds dried back when having reached the desiccation-sensitive state germinate poorly, if at all, and exhibit much reduced rates of protein synthesis in embryo tissue upon rehydration compared to desiccation-tolerant seeds. Embryo tissue from dried back, desiccation-tolerant seeds exhibits the capacity for much higher levels of protein synthesis than embryo tissue from unprimed seeds at equivalent stages of imbibition. RNA levels continue to increase in embryo tissue upon rehydration of dried back, desiccation-tolerant leek seeds but not in desiccation-sensitive seeds. Loss of protein synthetic capacity in desiccation-sensitive seeds during rehydration is accompanied by increasing levels of ribosomal RNA degradation. Such ribosomal RNA degradation may be indicative of the germinative capacity of leek seeds dried back after osmopriming treatments.

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Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing

10.21203/rs.3.rs-67621/v3 ◽

2021 ◽

Author(s):

Benjamin Kellman ◽

Hratch Baghdassarian ◽

Tiziano Pramparo ◽

Isaac Shamie ◽

Vahid Gazestani ◽

...

Keyword(s):

Differential Expression ◽

Rna Sequencing ◽

Ribosomal Rna ◽

Rna Degradation ◽

Library Preparation ◽

Rna Seq ◽

Rna Integrity ◽

Freeze Thaw ◽

The Impact ◽

Do So

Abstract Background: Both RNA-Seq and sample freeze-thaw are ubiquitous. However, knowledge about the impact of freeze-thaw on downstream analyses is limited. The lack of common quality metrics that are sufficiently sensitive to freeze-thaw and RNA degradation, e.g. the RNA Integrity Score, makes such assessments challenging.Results: Here we quantify the impact of repeated freeze-thaw cycles on the reliability of RNA-Seq by examining poly(A)-enriched and ribosomal RNA depleted RNA-seq from frozen leukocytes drawn from a toddler Autism cohort. To do so, we estimate the relative noise, or percentage of random counts, separating technical replicates. Using this approach we measured noise associated with RIN and freeze-thaw cycles. As expected, RIN does not fully capture sample degradation due to freeze-thaw. We further examined differential expression results and found that three freeze-thaws should extinguish the differential expression reproducibility of similar experiments. Freeze-thaw also resulted in a 3’ shift in the read coverage distribution along the gene body of poly(A)-enriched samples compared to ribosomal RNA depleted samples, suggesting that library preparation may exacerbate freeze-thaw-induced sample degradation.Conclusion: The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility.

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Computational discovery of hidden breaks in 28S ribosomal RNAs across eukaryotes and consequences for RNA Integrity Numbers

Scientific Reports ◽

10.1038/s41598-019-55573-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Paschalis Natsidis ◽

Philipp H. Schiffer ◽

Irepan Salvador-Martínez ◽

Maximilian J. Telford

Keyword(s):

Ribosomal Rna ◽

Convergent Evolution ◽

Animal Species ◽

Great Majority ◽

Computational Approach ◽

28S Rrna ◽

Rna Seq ◽

Ribosomal Rnas ◽

Rna Integrity ◽

Computational Discovery

AbstractIn some eukaryotes, a ‘hidden break’ has been described in which the 28S ribosomal RNA molecule is cleaved into two subparts. The break is common in protostome animals (arthropods, molluscs, annelids etc.), but a break has also been reported in some vertebrates and non-metazoan eukaryotes. We present a new computational approach to determine the presence of the hidden break in 28S rRNAs using mapping of RNA-Seq data. We find a homologous break is present across protostomes although it has been lost in a small number of taxa. We show that rare breaks in vertebrate 28S rRNAs are not homologous to the protostome break. A break is found in just 4 out of 331 species of non-animal eukaryotes studied and, in three of these, the break is located in the same position as the protostome break suggesting a striking instance of convergent evolution. RNA Integrity Numbers (RIN) rely on intact 28S rRNA and will be consistently underestimated in the great majority of animal species with a break.

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Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing

BMC Genomics ◽

10.1186/s12864-021-07381-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Benjamin P. Kellman ◽

Hratch M. Baghdassarian ◽

Tiziano Pramparo ◽

Isaac Shamie ◽

Vahid Gazestani ◽

...

Keyword(s):

Differential Expression ◽

Rna Sequencing ◽

Ribosomal Rna ◽

Rna Degradation ◽

Library Preparation ◽

Rna Seq ◽

Rna Integrity ◽

Freeze Thaw ◽

The Impact ◽

Do So

Abstract Background Both RNA-Seq and sample freeze-thaw are ubiquitous. However, knowledge about the impact of freeze-thaw on downstream analyses is limited. The lack of common quality metrics that are sufficiently sensitive to freeze-thaw and RNA degradation, e.g. the RNA Integrity Score, makes such assessments challenging. Results Here we quantify the impact of repeated freeze-thaw cycles on the reliability of RNA-Seq by examining poly(A)-enriched and ribosomal RNA depleted RNA-seq from frozen leukocytes drawn from a toddler Autism cohort. To do so, we estimate the relative noise, or percentage of random counts, separating technical replicates. Using this approach we measured noise associated with RIN and freeze-thaw cycles. As expected, RIN does not fully capture sample degradation due to freeze-thaw. We further examined differential expression results and found that three freeze-thaws should extinguish the differential expression reproducibility of similar experiments. Freeze-thaw also resulted in a 3′ shift in the read coverage distribution along the gene body of poly(A)-enriched samples compared to ribosomal RNA depleted samples, suggesting that library preparation may exacerbate freeze-thaw-induced sample degradation. Conclusion The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility. Graphical abstract

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Preservation of algal and higher plant ribosomal RNA integrity during extraction and electrophoretic quantitation

Analytical Biochemistry ◽

10.1016/0003-2697(78)90546-8 ◽

1978 ◽

Vol 91 (2) ◽

pp. 600-612 ◽

Cited By ~ 39

Author(s):

Lee McIntosh ◽

Rose Ann Cattolico

Keyword(s):

Ribosomal Rna ◽

Higher Plant ◽

Rna Integrity

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Insects’ RNA Profiling Reveals Absence of “Hidden Break” in 28S Ribosomal RNA Molecule of Onion Thrips,Thrips tabaci

Journal of Nucleic Acids ◽

10.1155/2015/965294 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

Rosaline Wanjiru Macharia ◽

Fidelis Levi Ombura ◽

Erick Onyango Aroko

Keyword(s):

Ribosomal Rna ◽

Thrips Tabaci ◽

28S Rrna ◽

Onion Thrips ◽

Rna Integrity ◽

Rna Profiling ◽

Insect Order ◽

Banding Profile ◽

Rrna Sequences

With an exception of aphids, insects’ 28S rRNA is thought to harbor a “hidden break” which cleaves under denaturing conditions to comigrate with 18S rRNA band to exhibit a degraded appearance on native agarose gels. The degraded appearance confounds determination of RNA integrity in laboratories that rely on gel electrophoresis. To provide guidelines for RNA profiles, RNA from five major insect orders, namely, Diptera, Hemiptera, Thysanoptera, Hymenoptera, and Lepidoptera, was compared under denaturing and nondenaturing conditions. This study confirmed that although present in most of insect’s RNA, the “hidden break” is absent in the 28S rRNA of onion thrips,Thrips tabaci. On the other hand, presence of “hidden break” was depicted in whiteflies’ 28S rRNA despite their evolutionary grouping under same order with aphids. Divergence of 28S rRNA sequences confirms variation of both size and composition of gap region among insect species. However, phylogeny reconstruction does not support speciation as a possible source of the hidden break in insect’s 28S rRNA. In conclusion, we show that RNA from a given insect order does not conform to a particular banding profile and therefore this approach cannot be reliably used to characterize newly discovered species.

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Differential amplicons for the evaluation of RNA integrity extracted from complex environmental samples

10.1101/401109 ◽

2018 ◽

Author(s):

Fabien Cholet ◽

Umer Z. Ijaz ◽

Cindy J. Smith

Keyword(s):

Community Structure ◽

16S Rrna ◽

Ribosomal Rna ◽

Amplicon Sequencing ◽

Rna Degradation ◽

Validity Of Results ◽

Rna Integrity ◽

Rna Integrity Number ◽

Degraded Samples ◽

Integrity Index

AbstractBackgroundReliability and reproducibility of transcriptomics-based studies are highly dependent on the integrity of RNA. Microfluidics-based techniques based on ribosomal RNA such as the RNA Integrity Number (RIN) are currently the only approaches to evaluate RNA integrity. However, it is not known if ribosomal RNA reflects the integrity of the meaningful part of the sample, the mRNA. Here we test this assumption and present a new integrity index, the Ratio amplicon, Ramp, to monitor mRNA integrity based on the differential amplification of long to short RT-Q-PCR amplicons of the glutamine synthetase A (glnA) transcript.ResultsWe successfully designed and tested two Rampindexes targetingglnAtranscripts. We showed in a suite of experimental degradations of RNA extracted from sediment that while the RIN in general did reflect the degradation status of the RNA well the Rampmapped mRNA degradation better as reflected by changes in Reverse Transcriptase Quantitative PCR (RT-Q-PCR) results. Furthermore, we examined the effect of degradation on transcript community structure by amplicon sequencing of the16S rRNA, amoAandglnAtranscript which was successful even form the highly-degraded samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed between successively degraded 16S rRNA transcripts profiles.ConclusionAs demonstrated, transcripts can be quantified and sequenced even from highly degraded samples. Therefore, we strongly recommend that a quality check of RNA is conducted to ensure validity of results. For this both the RIN and Rampare useful, with the Rampbetter evaluating mRNA integrity in this study.

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Computational discovery of hidden breaks in 28S ribosomal RNAs across eukaryotes and consequences for RNA Integrity Numbers

10.1101/773226 ◽

2019 ◽

Author(s):

Paschalis Natsidis ◽

Philipp H. Schiffer ◽

Irepan Salvador-Martínez ◽

Maximilian J. Telford

Keyword(s):

Ribosomal Rna ◽

Convergent Evolution ◽

Animal Species ◽

Great Majority ◽

Computational Approach ◽

28S Rrna ◽

Rna Seq ◽

Ribosomal Rnas ◽

Rna Integrity ◽

Computational Discovery

ABSTRACTIn some eukaryotes, a ‘hidden break’ has been described in which the 28S ribosomal RNA molecule is cleaved into two subparts. The break is common in protostome animals (arthropods, molluscs, annelids etc.) but a break has also been reported in some vertebrates and non-metazoan eukaryotes. We present a new computational approach to determine the presence of the hidden break in 28S rRNAs using mapping of RNA-Seq data. We find a homologous break is present across protostomes although has been lost in a small number of taxa. We show that rare breaks in vertebrate 28S rRNAs are not homologous to the protostome break. A break is found in just 4 out of 331 species of non-animal eukaryotes studied and three of these are located in the same position as the protostome break suggesting a striking instance of convergent evolution. RNA Integrity Numbers (RIN) rely on intact 28s rRNA and will be consistently underestimated in the great majority of animal species with a break.

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