A new reference gene, Ef1A, for quantitative real-time PCR assay of the starfish Asterias rubens pyloric ceca

Садритдинова, А. Ф.; Снежкина, А. В.; Dmitriev, Alexey A.; Krasnov, George S.; Astakhova, L. N.; Kudryavtsev, A. A.; Melnikova, N. V.; Speranskaya, A.S.; Дарий, М. В.; Lakunina, V. A.; Урошлев, Л. А.; Smurov, A O; Степанов, О. А.; Kudryavtseva, Anna V.

doi:10.1134/s0012496613050050

Cited by 9 publications

(7 citation statements)

References 9 publications

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“…Primer pairs used for amplifying PpCT-precursor, with elongation factor 1a (EF1α) (accession no. : GFOQ01387693) cDNA as a control for normalization (Sadritdinova et al, 2013 ), were PpCT qPCR-F and -R and EF1α qPCR-F and -R, respectively (Supplemental Table 2 ). The thermal-cycling profile was 95°C for 10 min, followed by 40 cycles at 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s, with fluorescence recording at the end of each cycle.…”

Section: Methodsmentioning

confidence: 99%

Biochemical, Anatomical, and Pharmacological Characterization of Calcitonin-Type Neuropeptides in Starfish: Discovery of an Ancient Role as Muscle Relaxants

Cai

Kim

et al. 2018

Front. Neurosci.

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Calcitonin (CT) is a peptide hormone released by the thyroid gland that regulates blood Ca2+ levels in mammals. The CT gene is alternatively spliced, with one transcript encoding CT and another transcript encoding the CT-like neuropeptide calcitonin-gene related peptide (α-CGRP), which is a powerful vasodilator. Other CT-related peptides in vertebrates include adrenomedullin, amylin, and intermedin, which also act as smooth muscle relaxants. The evolutionary origin of CT-type peptides has been traced to the bilaterian common ancestor of protostomes and deuterostomes and a CT-like peptide (DH31) has been identified as a diuretic hormone in some insect species. However, little is known about the physiological roles of CT-type peptides in other invertebrates. Here we characterized a CT-type neuropeptide in a deuterostomian invertebrate—the starfish Asterias rubens (Phylum Echinodermata). A CT-type precursor cDNA (ArCTP) was sequenced and the predicted structure of the peptide (ArCT) derived from ArCTP was confirmed using mass spectrometry. The distribution of ArCTP mRNA and the ArCT peptide was investigated using in situ hybridization and immunohistochemistry, respectively, revealing stained cells/processes in the nervous system, digestive system, and muscular organs, including the apical muscle and tube feet. Investigation of the effects of synthetic ArCT on in vitro preparations of the apical muscle and tube feet revealed that it acts as a relaxant, causing dose-dependent reversal of acetylcholine-induced contraction. Furthermore, a muscle relaxant present in whole-animal extracts of another starfish species, Patiria pectinifera, was identified as an ortholog of ArCT and named PpCT. Consistent with the expression pattern of ArCTP in A. rubens, RT-qPCR revealed that in P. pectinifera the PpCT precursor transcript is more abundant in the radial nerve cords than in other tissues/organs analyzed. In conclusion, our findings indicate that the physiological action of CT-related peptides as muscle relaxants in vertebrates may reflect an evolutionarily ancient role of CT-type neuropeptides that can be traced back to the common ancestor of deuterostomes.

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Section: Methodsmentioning

confidence: 99%

Biochemical, Anatomical, and Pharmacological Characterization of Calcitonin-Type Neuropeptides in Starfish: Discovery of an Ancient Role as Muscle Relaxants

Cai

Kim

et al. 2018

Front. Neurosci.

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“…Reference genes are considered to be those with constant expression in all samples of the organism, including different tissues, or under different treatments, or at different developmental stages. Previous studies have shown that the common reference genes in many plants are those coding for elongation factor 1-α ( EF-1α ) 13 , actin ( ACT ) 14 , glyceraldehyde-3-phosphate dehydrogenase ( GADPH ) 15 , and 18S ribosomal RNA ( 18S ) 16 , to name a few. However, it can be seen from previous studies that there is no universal reference gene across different species, and the most stable reference genes are different in different plants or under different treatments of the same plant 8 .…”

Section: Introductionmentioning

confidence: 99%

Validation of suitable reference genes by various algorithms for gene expression analysis in Isodon rubescens under different abiotic stresses

Lian

Bao

Yang

et al. 2022

Sci Rep

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Isodon rubescens (Hemsley) H. Hara (Lamiaceae) is a traditional Chinese medicine plant that has been used to treat various human diseases. Oridonin is one of the main active ingredients, and the route of its molecular biosynthesis remains to be determined. The study of gene expression patterns can provide clues toward the understanding of its biological functions. The selection of suitable reference genes for normalizing target gene expression is the first steps in any quantitative real-time PCR (RT-qPCR) gene expression study. Therefore, validation of suitable reference genes is necessary for obtaining reliable results in RT-qPCR analyses of I. rubescens. Here, 12 candidate reference genes were chosen, and their expression stability in different tissues of I. rubescens and in leaves under different abiotic stresses (NaCl, dehydration, SA, MeJA, and ABA) was evaluated using the ∆Ct, NormFinder, GeNorm, BestKeeper, and RankAggreg statistical tools. Analysis using the comprehensive tools of RankAggreg algorithm showed that GADPH, 18S and eIF were stably expressed in different tissues; UBQ, Apt, and HIS; Cycl, UBQ, and PP2A; GADPH, 18S, and eIF; eIF, UBQ, and PP2A; TUB, Cycl, and UBQ; were the best three candidate reference genes for the samples of Dehydration, NaCl, SA, MeJA, and ABA treatment, respectively. While for the concatenated sets of ND (NaCl and dehydration) and SMA (SA, MeJA, and ABA), UBQ, HIS, and TUA; UBQ, eIF and Apt were the three appropriate candidate reference genes, respectively. In addition, the expression patterns of HMGR in different tissues and under different treatments were used to confirm the reliability of the selected reference genes, indicating that the use of an inappropriate reference gene as the internal control will cause results with a large deviation. This work is the first study on the expression stability of reference genes in I. rubescens and will be particularly useful for gene functional research in this species.

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“…In shrimp, genes such as β‐actin , glyceraldehyde‐3 phosphate dehydrogenase ( GAPDH ), elongation factor‐1 ( EF1‐α ), 18S ribosomal RNA ( 18S rRNA ) and 7S ribosomal RNA ( 7S rRNA ) are commonly used as reference genes in a variety of experimental conditions (Alvarez‐Lee et al, 2020; Dhar et al, 2009; Hu et al, 2018; Jorgensen et al, 2006; Leelatanawit et al, 2012; Pan et al, 2015). The expression stability of reference genes may vary depending on tissues, developmental stage, or environmental conditions; in other words, there is no universal reference gene that maintains stable expression in all tissues and conditions (Hu et al, 2018; Sadritdinova et al, 2013). Bustin et al (2009) published guidelines describing the minimum information necessary for evaluating RT‐qPCR experiments.…”

Section: Introductionmentioning

confidence: 99%

Validation of reference genes for gene expression analysis in melanin‐injected black tiger shrimp ( Penaeus monodon )

Phan

Nguyen

et al. 2022

Aquaculture Research

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The expression stability of four potential reference genes (EF1α, β-actin, GAPDH and SubF0) was evaluated to normalize the expression of six immune-related genes (proPO1, proPO2, MnSOD, PAP, Rab7 and NOS) in different tissues of black tiger shrimp (Penaeus monodon) following melanin injection and using the geNorm and NormFinder algorithms. The stability rankings of the reference genes were as follows: EF1α > βactin > SubF0 > GAPDH in the muscle, GAPDH > EF1α > SubF0 > β-actin in the carapace and EF1α > GAPDH > β-actin > SubF0 in the haemolymph. To normalize the expression of six immune-related genes of P. monodon in different tissues under the effect of melanin injection, EF1α and GAPDH alone were the most suitable reference genes for the muscle and carapace, respectively, whereas the combination of EF1α + β-actin was the most suitable for the haemolymph. The expression of these six immunerelated genes in P. monodon was upregulated in the haemolymph, downregulated in the carapace and nearly unchanged in the muscle of the melanin-injected group compared to that in the control group. Our results provide reference genes for reverse transcription quantitative polymerase chain reaction to analyse the protective effects of melanin in black tiger shrimp.

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A new reference gene, Ef1A, for quantitative real-time PCR assay of the starfish Asterias rubens pyloric ceca

Cited by 9 publications

References 9 publications

Biochemical, Anatomical, and Pharmacological Characterization of Calcitonin-Type Neuropeptides in Starfish: Discovery of an Ancient Role as Muscle Relaxants

Biochemical, Anatomical, and Pharmacological Characterization of Calcitonin-Type Neuropeptides in Starfish: Discovery of an Ancient Role as Muscle Relaxants

Validation of suitable reference genes by various algorithms for gene expression analysis in Isodon rubescens under different abiotic stresses

Validation of reference genes for gene expression analysis in melanin‐injected black tiger shrimp ( Penaeus monodon )

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