Evaluation of Suitable Reference Genes for Normalization of RT-qPCR in Echiura (Urechis unicinctus) during Developmental Process

Wei, Maokai; Lu, Li; Wang, Qingchao; Kong, Dexu; Zhang, T.; Qin, Zhenkui; Zhang, Z.

doi:10.1134/s1063074019300023

Cited by 12 publications

(5 citation statements)

References 15 publications

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“…The qRT-PCR condition was: denature at 95 °C for 30 s, followed by 39 cycles of 5 s at 95 °C, and 60 °C for 30 s. Each sample was run in 3 technical replicates. The relative expression levels were normalized to the reference gene ATPase [ 80 ], and expression ratios were calculated using the 2 –ΔΔCt method. The experimental data were presented as mean ± standard deviation from three samples with three parallel repetitions, and all RT-PCR assays were validated in compliance with “the MIQE guidelines” [ 81 ].…”

Section: Methodsmentioning

confidence: 99%

Identification of the neuropeptide precursor genes potentially involved in the larval settlement in the Echiuran worm Urechis unicinctus

et al. 2020

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Background In marine invertebrate life cycles, which often consist of planktonic larval and benthonic adult stages, settlement of the free-swimming larva to the sea floor in response to environmental cues is a key life cycle transition. Settlement is regulated by a specialized sensory–neurosecretory system, the larval apical organ. The neuroendocrine mechanisms through which the apical organ transduces environmental cues into behavioral responses during settlement are not fully understood yet. Results In this study, a total of 54 neuropeptide precursors (pNPs) were identified in the Urechis unicinctus larva and adult transcriptome databases using local BLAST and NpSearch prediction, of which 10 pNPs belonging to the ancient eumetazoa, 24 pNPs belonging to the ancient bilaterian, 3 pNPs belonging to the ancient protostome, 9 pNPs exclusive in lophotrochozoa, 3 pNPs exclusive in annelid, and 5 pNPs only found in U. unicinctus. Furthermore, four pNPs (MIP, FRWamide, FxFamide and FILamide) which may be associated with the settlement and metamorphosis of U. unicinctus larvae were analysed by qRT-PCR. Whole-mount in situ hybridization results showed that all the four pNPs were expressed in the region of the apical organ of the larva, and the positive signals were also detected in the ciliary band and abdomen chaetae. We speculated that these pNPs may regulate the movement of larval cilia and chaeta by sensing external attachment signals. Conclusions This study represents the first comprehensive identification of neuropeptides in Echiura, and would contribute to a complete understanding on the roles of various neuropeptides in larval settlement of most marine benthonic invertebrates.

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Section: Methodsmentioning

confidence: 99%

Identification of the neuropeptide precursor genes potentially involved in the larval settlement in the Echiuran worm Urechis unicinctus

et al. 2020

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“…The hierarchical clustering of the pNP genes according to their FPKM values in the U. unicinctus larval transcriptome [59] was performed by an online tool (https://www.omicshare.com/tools/). [74], and expression ratios were calculated using the 2 -ΔΔCt method. The experimental data were presented as mean ± standard deviation from three samples with three parallel repetitions, and all RT-PCR assays were validated in compliance with "the MIQE guidelines" [75].…”

Section: Identification Functional Annotation and Sequence Alignmentmentioning

confidence: 99%

Identification of the Neuropeptide Precursor Genes Potentially Involved in the Larval Settlement in the Echiuran Worm Urechis unicinctus

Hou

Qin

Wei

et al. 2020

Preprint

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Background: In marine invertebrate life cycles, which often consist of planktonic larval and benthonic adult stages, settlement of the free-swimming larva to the sea ﬂoor in response to environmental cues is a key life cycle transition. Settlement is regulated by a specialized sensory–neurosecretory system, the larval apical organ. The neuroendocrine mechanisms through which the apical organ transduces environmental cues into behavioral responses during settlement are not fully understood yet.Results: In this study, a total of 54 neuropeptide precursors (pNPs) were identified in the Urechis unicinctus larva and adult transcriptome databases using local BLAST and de novo prediction, of which 10 pNPs belonging to the ancient eumetazoa, 23 pNPs belonging to the ancient bilaterian, 3 pNPs belonging to the ancient protostome, 10 pNPs exclusive in lophotrochozoa, 3 pNPs exclusive in annelid, and 5 pNPs only found in U. unicinctus. Furthermore, four pNPs (MIP, FRWamide, FxFamide and FILamide) which may be associated with the settlement and metamorphosis of U. unicinctus larvae were verified successfully by qRT-PCR. Whole-mount in situ hybridization results showed that all the four pNPs were expressed in the region of the apical organ of the larva, and the positive signals were also detected in the ciliary band and abdomen chaetae. We speculated that these pNPs may regulate the movement of larval cilia and chaeta by sensing external attachment signals.Conclusions: This study represents the ﬁrst comprehensive identiﬁcation of neuropeptides in Echiura, and would contribute to a complete understanding on the roles of various neuropeptides in larval settlement of most marine benthonic invertebrates.

show abstract

“…The qRT-PCR condition was: denature at 95 ℃ for 30 s, followed by 39 cycles of 5 s at 95 ℃, and 60 ℃ for 30 s. Each sample was run in 3 technical replicates. The relative expression levels were normalized to the reference gene ATPase [80], and expression ratios were calculated using the 2 -ΔΔCt method. The experimental data were presented as mean ± standard deviation from three samples with three parallel repetitions, and all RT-PCR assays were validated in compliance with "the MIQE guidelines" [81].…”

Section: Identification Classi Cation and Sequence Alignment Of Neurmentioning

confidence: 99%

Identification of the Neuropeptide Precursor Genes Potentially Involved in the Larval Settlement in the Echiuran Worm Urechis unicinctus

Hou

Qin

Wei

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: In marine invertebrate life cycles, which often consist of planktonic larval and benthonic adult stages, settlement of the free-swimming larva to the sea ﬂoor in response to environmental cues is a key life cycle transition. Settlement is regulated by a specialized sensory–neurosecretory system, the larval apical organ. The neuroendocrine mechanisms through which the apical organ transduces environmental cues into behavioral responses during settlement are not fully understood yet.Results: In this study, a total of 54 neuropeptide precursors (pNPs) were identified in the Urechis unicinctus larva and adult transcriptome databases using local BLAST and NpSearch prediction, of which 10 pNPs belonging to the ancient eumetazoa, 24 pNPs belonging to the ancient bilaterian, 3 pNPs belonging to the ancient protostome, 9 pNPs exclusive in lophotrochozoa, 3 pNPs exclusive in annelid, and 5 pNPs only found in U. unicinctus. Furthermore, four pNPs (MIP, FRWamide, FxFamide and FILamide) which may be associated with the settlement and metamorphosis of U. unicinctus larvae were analysed by qRT-PCR. Whole-mount in situ hybridization results showed that all the four pNPs were expressed in the region of the apical organ of the larva, and the positive signals were also detected in the ciliary band and abdomen chaetae. We speculated that these pNPs may regulate the movement of larval cilia and chaeta by sensing external attachment signals.Conclusions: This study represents the ﬁrst comprehensive identiﬁcation of neuropeptides in Echiura, and would contribute to a complete understanding on the roles of various neuropeptides in larval settlement of most marine benthonic invertebrates.

show abstract

Evaluation of Suitable Reference Genes for Normalization of RT-qPCR in Echiura (Urechis unicinctus) during Developmental Process

Cited by 12 publications

References 15 publications

Identification of the neuropeptide precursor genes potentially involved in the larval settlement in the Echiuran worm Urechis unicinctus

Identification of the neuropeptide precursor genes potentially involved in the larval settlement in the Echiuran worm Urechis unicinctus

Identification of the Neuropeptide Precursor Genes Potentially Involved in the Larval Settlement in the Echiuran Worm Urechis unicinctus

Identification of the Neuropeptide Precursor Genes Potentially Involved in the Larval Settlement in the Echiuran Worm Urechis unicinctus

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