High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study

Asai, Sneha; Ianora, Adrianna; Lauritano, Chiara; Lindeque, Penelope K.; Carotenuto, Ylenia

doi:10.1016/j.margen.2014.12.004

Cited by 25 publications

(23 citation statements)

References 19 publications

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“…Finally, we assessed RNA integrity from laser-microdissected intestine and non-intestine from magnesium-treated, methacarn-fixed tissue (Fig S1D, Fig 1A and 1B), stained only with Eosin Y to further minimize processing time (Fig S1C). Although additional freezing, cryosectioning, staining, and drying steps required for LCM caused a modest decrease in RNA integrity relative to whole animals (compare Fig S1A and Fig S1D), prominent 18S/28S rRNA peaks (which co-migrate in planarians, as in some other invertebrates (54–57)) (Fig S1D) indicated that the combination of magnesium-induced relaxation, brief methacarn fixation, and rapid Eosin Y staining were suitable for LCM and extraction of RNA of sufficient quality for RNA-Seq.…”

Section: Resultsmentioning

confidence: 99%

“… (A) RNA yields (left table) and Bioanalyzer/TapeStation analysis of RNA integrity (right panels) from whole planarian samples (15 planarians, ∼20-25 mg total tissue) treated as shown. As in some other invertebrates (54–57), heat denaturation prior to analysis causes 28S rRNA to co-migrate as two bands with 18S rRNA (indicated with red arrows). Samples are representative of at least three independent replicates.…”

Section: Supplementary Materials Legendsmentioning

confidence: 95%

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Cell-type diversity and regionalized gene expression in the planarian intestine revealed by laser-capture microdissection transcriptome profiling

Forsthoefel

Cejda

Khan

et al. 2019

Preprint

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Organ regeneration requires precise coordination of new cell differentiation and remodeling of uninjured tissue to faithfully re-establish organ morphology and function. An atlas of gene expression and cell types in the uninjured state is therefore an essential pre-requisite for understanding how damage is repaired. Here, we use laser-capture microdissection (LCM) and RNA-Seq to define the transcriptome of the intestine of Schmidtea mediterranea, a planarian flatworm with exceptional regenerative capacity. Bioinformatic analysis of 1,844 intestine-enriched transcripts suggests extensive conservation of digestive physiology with other animals, including humans. Comparison of the intestinal transcriptome to purified absorptive intestinal cell (phagocyte) and published single-cell expression profiles confirms the identities of known intestinal cell types, and also identifies hundreds of additional transcripts with previously undetected intestinal enrichment. Furthermore, by assessing the expression patterns of 143 transcripts in situ, we discover unappreciated mediolateral regionalization of gene expression and cell-type diversity, especially among goblet cells. Demonstrating the utility of the intestinal transcriptome, we identify 22 intestine-enriched transcription factors, and find that several have distinct functional roles in the regeneration and maintenance of goblet cells. Furthermore, depletion of goblet cells inhibits planarian feeding and reduces viability. Altogether, our results show that LCM is a viable approach for assessing tissue-specific gene expression in planarians, and provide a new resource for further investigation of digestive tract regeneration, the physiological roles of intestinal cell types, and axial polarity.

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Section: Resultsmentioning

confidence: 99%

Section: Supplementary Materials Legendsmentioning

confidence: 95%

Cell-type diversity and regionalized gene expression in the planarian intestine revealed by laser-capture microdissection transcriptome profiling

Forsthoefel

Cejda

Khan

et al. 2019

Preprint

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“…This difference in resistance, and other physiological differences associated with reproduction may result in different transcriptional responses to stressors. When studying adult copepods, the selection of sex or the choice to include a mixture of sexes, should be carefully considered in light of the scientific Asai et al, 2015;Zhang et al, 2013). While DNAse is frequently used during the…”

Section: Selection Of Developmental Stagementioning

confidence: 99%

“…In automated electrophoresis of total RNA, the most commonly used single metric of quality in the RNA Integrity Number (RIN), which is derived from an algorithm that compares the relative proportions of 28S and 18S rRNA; values range from 1 to 10, with lower values indicating degradation. In copepods, like many other arthropods, the 28S band is fragile and can break ("the hidden break") during sample preparation (Asai et al, 2015;McCarthy et al, 2015). Thus, many copepod studies disregard the RIN metrics and rely on a subjective visual analysis of an electropherogram trace, including the presence of a strong discrete 18S band, the absence of larger bands indicating contamination by genomic DNA, and limited smearing within the smaller size ranges (e.g., Almada andTarrant 2016, Zhou et al 2018).…”

Section: Selection Of Developmental Stagementioning

confidence: 99%

“…Thus, many copepod studies disregard the RIN metrics and rely on a subjective visual analysis of an electropherogram trace, including the presence of a strong discrete 18S band, the absence of larger bands indicating contamination by genomic DNA, and limited smearing within the smaller size ranges (e.g., Almada andTarrant 2016, Zhou et al 2018). In many cases 28S breakage occurs during heat denaturing of samples immediately before analysis, so integrity can sometimes be preserved by omitting this step (e.g, Figure 2 within Asai et al 2015).…”

Section: Selection Of Developmental Stagementioning

confidence: 99%

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Molecular physiology of copepods - from biomarkers to transcriptomes and back again

Tarrant

Nilsson

Hansen

2019

Comparative Biochemistry and Physiology Part D: Genomics and Pr

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Planktonic copepods are a diverse and abundant group of small (⁓mm sized) aquatic animals that play a critical role in linking the base of the food chain with higher trophic levels. These invertebrates are a primary food source for marine fish larvae. Their ubiquitous presence is thus of vital importance for recruitment of fish stocks and also as promising live feed for finfish production in aquaculture. This paper reviews the application of molecular approaches to understanding copepod physiology, particularly in non-parasitic species. The review includes both targeted gene approaches and untargeted transcriptomic approaches, with suggestions for best practices in each case. Issues particularly relevant to studies of copepods include heterogeneity within species, morphologically cryptic species, experimental artifacts associated with sample handling, and limited annotation of copepod genes and transcripts. The emergence of high-throughput sequencing and associated increased availability of genomic and transcriptomic databases has presented a huge opportunity to advance knowledge of copepod physiology. The research community can leverage this opportunity through efforts to maintain or improve data accessibility, database annotation, and documentation of analytical pipelines.

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Annual transcriptome of a key zooplankton species, the copepod Calanus finmarchicus

Payton

Noirot

Last

et al. 2022

Ecology and Evolution

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The copepod Calanus finmarchicus (Crustacea, Copepoda) is a key zooplanktonic species with a crucial position in the North Atlantic food web and significant contributor to ocean carbon flux. Like many other high latitude animals, it has evolved a programmed arrested development called diapause to cope with long periods of limited food supply, while growth and reproduction are timed to take advantage of seasonal peaks in primary production. However, anthropogenic warming is inducing changes in the expected timing of phytoplankton blooms, suggesting phenological mismatches with negative consequences for the N. Atlantic ecosystem. While diapause mechanisms are mainly studied in terrestrial arthropods, specifically on laboratory model species, such as the fruit fly Drosophila, the molecular investigations of annual rhythms in wild marine species remain fragmentary. Here we performed a rigorous year‐long monthly sampling campaign of C. finmarchicus in a Scottish Loch (UK; 56.45°N, 5.18°W) to generate an annual transcriptome. The mRNA of 36 samples (monthly triplicate of 25 individuals) have been deeply sequenced with an average depth of 137 ± 4 million reads (mean ± SE) per sample, aligned to the reference transcriptome, and filtered. We detail the quality assessment of the datasets and provide a high‐quality resource for the investigation of wild annual transcriptomic rhythms (35,357 components) in a key diapausing zooplanktonic species.

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High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study

Cited by 25 publications

References 19 publications

Cell-type diversity and regionalized gene expression in the planarian intestine revealed by laser-capture microdissection transcriptome profiling

Cell-type diversity and regionalized gene expression in the planarian intestine revealed by laser-capture microdissection transcriptome profiling

Molecular physiology of copepods - from biomarkers to transcriptomes and back again

Annual transcriptome of a key zooplankton species, the copepod Calanus finmarchicus

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