Selection of reference genes as internal controls for gene expression in tissues of red abalone Haliotis rufescens (Mollusca, Vetigastropoda; Swainson, 1822)

López-Landavery, Edgar A.; Portillo-López, Amelia; Gallardo‐Escárate, Cristian; Río-Portilla, Miguel A. Del

doi:10.1016/j.gene.2014.08.002

Cited by 15 publications

(9 citation statements)

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“…We examined different salinity stress levels, skin muscle injury, different tissues of O. reevesii adults and individuals with different weights to identify reference genes. The most suitable internal reference genes in different tissues of Haliotis rufescens are reportedly RPL5S and CYC; the most stable in O. reevesii was found to be the RPL28S 36 , but EF1a can serve as an alternative. When analysing differences in target gene expression in various tissues of O. reevesii, RPL28S should be used as the reference gene if the abundance of target gene expression is low, whereas EF1a is recommended if target gene expression is high.…”

Section: Discussionmentioning

confidence: 99%

Peer Review #1 of "Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii (v0.1)"

Schnytzer¹

2019

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Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there have been few analyses of stable housekeeping genes in Onchidium reevesii under salinity stress and injury. In this study, the gene expression stabilities of seven commonly used housekeeping genes, CYC, RPL28S, ACTB, TUBB, EF1a, Ubiq and 18S RNA, were investigated using BestKeeper, geNorm, NormFinder and RefFinfer. Although the results of the four programs varied to some extent, in general, RPL28S, TUBB, ACTB and EF1a were ranked highly. ACTB and TUBB were found to be the most stable housekeeping genes under salinity stress, and EF1a plus TUBB was the most stable combination under injury stress. When analysing target gene expression in different tissues, RPL28S or EF1a should be selected as the reference gene according to the level of target gene expression. Under extreme environmental stress (salinity) conditions, ACTB (0 ppt, 5 ppt, 15 ppt, 25 ppt) and TUBB (35 ppt) are reasonable reference gene choices when expression stability and abundance are considered. Under conditions of 15 ppt salinity and injury stress, our results showed that the best two-gene combination was TUBB plus EF1a. Therefore, we suggest that RPL28S, ACTB and TUBB are suitable reference genes for evaluating mRNA transcript levels. Based on candidate gene expression analysis, the tolerance of O. reevesii to low salinity (low osmotic pressure) is reduced compared to its tolerance to high salinity (high osmotic pressure). These findings will help researchers obtain accurate results in future quantitative gene expression analyses of O. reevesii under other stress conditions.

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Section: Discussionmentioning

confidence: 99%

Peer Review #1 of "Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii (v0.1)"

Schnytzer¹

2019

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“…RL5 and RL28 belong to the large subunit ribosomal protein family, while RS8 and RS25 belong to the small subunit family, and they are present in all cell types involved in biogenesis of new proteins. Studies found that RL5 was the most stable gene in all tissues in red abalone (López-Landavery et al, 2014) and in disk abalone following exposure to tributyltinchloride and 17 β -estradiol (Wan et al, 2011). The other genes relating to ribosomal protein biosynthesis are commonly considered as housekeeping genes in many other organisms, including animals, plants, and algae (Barsalobres-Cavallari et al, 2009; Hsiao et al, 2001; Liu et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa)

Song

Dang

et al. 2017

PeerJ

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BackgroundThe veined rapa whelk Rapana venosa is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in R. venosa. For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes in R. venosa for use as internal controls for qRT-PCR.MethodsIn this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1α (EF-1α), α-actin (ACT), cytochrome c oxidase subunit 1 (COX1), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1α subcomplex subunit 7 (NDUFA7), 60S ribosomal protein L5 (RL5), 60S ribosomal protein L28 (RL28), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-tubulin (TUBB), 40S ribosomal protein S25 (RS25), 40S ribosomal protein S8 (RS8), ubiquitin-conjugating enzyme E2 (UBE2), histone H3 (HH3), and peptidyl-prolyl cis-trans isomerase A (PPIA). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms.ResultsOf the 13 candidate genes tested, we found that EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. For the normalization of a single specific tissue, we suggested that EF-1α and NDUFA7 are the best combination in gonad, as well as COX1 and RL28 for intestine, EF-1α and RL5 for kidney, EF-1α and COX1 for gill, EF-1α and RL28 for Leiblein and mantle, EF-1α, RL5, and NDUFA7 for liver, GAPDH, PPIA, and RL28 for hemocyte. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured, and COX1 and RL5 were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization, PPIA, RS25, and RL28 for stage 1, RL5 and RL28 for stage 2 and 5, RL28 and NDUFA7 for stage 3, and PPIA and TUBB for stage 4.DiscussionOur results are instrumental for the selection of appropriately validated housekeeping genes for use as internal controls for gene expression studies in adult tissues or larval development of R. venosa in the future.

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“…In H. discus discus, GAPDH was deemed as an unreliable internal control (Wan et al, 2011) while in Haliotis rufescens GAPDH suitability depended on the type of tissue subject to analysis (López-Landavery et al, 2014). GAPDH is a tetrameric enzyme that converts glucose into pyruvic acid in the glycolytic pathway.…”

Section: Glyceraldehyde-3-phosphate Dehydrogenase (Gapdh)mentioning

confidence: 99%

Identification of differentially expressed reproductive and metabolic proteins in the female abalone ( Haliotis laevigata ) gonad following artificial induction of spawning

Mendoza-Porras

Botwright

Reverter

et al. 2017

Comparative Biochemistry and Physiology Part D: Genomics and Pr

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Selection of reference genes as internal controls for gene expression in tissues of red abalone Haliotis rufescens (Mollusca, Vetigastropoda; Swainson, 1822)

Cited by 15 publications

References 60 publications

Peer Review #1 of "Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii (v0.1)"

Peer Review #1 of "Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii (v0.1)"

Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa)

Identification of differentially expressed reproductive and metabolic proteins in the female abalone ( Haliotis laevigata ) gonad following artificial induction of spawning

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