Validation of housekeeping genes for gene expression studies in an ice alga Chlamydomonas during freezing acclimation

Liu, Chenlin; Wu, Guangting; Huang, Xiaohang; Liu, Shenghao; Cong, Bin

doi:10.1007/s00792-012-0441-4

Cited by 46 publications

(34 citation statements)

References 34 publications

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“…showed that gapdh was an unsuitable HKG for normalising gene expression in thermal and light studies. Nevertheless, gapdh is the most stable HKG in ice alga Chlamydomonas in freezing acclimation studies44, which is consistent with the results of the present study, indicating the stability of gapdh mRNA during low-temperature treatment. Moreover, gapdh was found to be a suitable HKG for normalising gene expression in red alga Porphyra yezoensis at various life stages24.…”

Section: Discussionsupporting

confidence: 93%

Validation of reference genes for cryopreservation studies with the gorgonian coral endosymbiont Symbiodinium

Chong

Kuo

Tsai

et al. 2017

Sci Rep

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Quantification by real-time RT-PCR requires a stable internal reference known as a housekeeping gene (HKG) for normalising the mRNA levels of target genes. The present study identified and validated stably expressed HKGs in post-thaw Symbiodinium clade G. Six potential HKGs, namely, pcna, gapdh, 18S rRNA, hsp90, rbcl, and ps1, were analysed using three different algorithms, namely, GeNorm, NormFinder, and BestKeeper. The GeNorm algorithm ranked the candidate genes as follows in the order of decreasing stability: pcna and gapdh > ps1 > 18S rRNA > hsp90 > rbcl. Results obtained using the NormFinder algorithm also showed that pcna was the most stable HKG and ps1 was the second most stable HKG. We found that the candidate HKGs examined in this study showed variable stability with respect to the three algorithms. These results indicated that both pcna and ps1 were suitable for normalising target gene expression determined by performing real-time RT-PCR in cryopreservation studies on Symbiodinium clade G. The results of the present study would help future studies to elucidate the effect of cryopreservation on gene expression in dinoflagellates.

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Section: Discussionsupporting

confidence: 93%

Validation of reference genes for cryopreservation studies with the gorgonian coral endosymbiont Symbiodinium

Chong

Kuo

Tsai

et al. 2017

Sci Rep

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“…(Cao et al 2012), Chlamydomonas sp. ICE-L (Liu et al 2012), the dinoflagellate Prorocentrum minimum (Guo and Ki 2012a, b), and the diatoms Ditylum brightwellii (Guo et al 2013), Skeletonema costatum, Chaetoceros affinis (Kang et al 2012), Talasiossira pseudonana (Alexander et al 2012), and Pseudo-nitzschia multistriata (Adelfi et al 2014).…”

Section: Resultsmentioning

confidence: 99%

“…In the present study, we selected putative reference genes by considering those reported in previous studies ( Vandesompele et al 2002;Reid et al 2006;Silver et al 2006;Liu et al 2012) as well as the available genes from our EST data. We selected nine HKGs, that is, 18S rRNA (18S), actin (ACT), alpha tubulin (TUA), beta tubulin (TUB), ubiquitin (UBQ), ribosomal protein s4 (rps4), eukaryotic initiation factor (eIF), histone H4 (H4), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as candidate internal reference genes for the green alga C. ehrenbergii.…”

Section: Housekeeping Gene Selection and Primer Designmentioning

confidence: 99%

Evaluation and selection of reference genes for ecotoxicogenomic study of the green alga Closterium ehrenbergii using quantitative real-time PCR

et al. 2015

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The green alga Closterium ehrenbergii occurs in fresh water environments and has been suggested as a model for ecotoxicological assessment. Quantitative real-time PCR (qRT-PCR), with its high sensitivity and specificity, is a preferred method for reliable quantification of gene expression levels. qRT-PCR requires reference genes to normalize the transcription level of the target gene, and selection of appropriate references is crucial. Here, we evaluated nine housekeeping genes, that is, 18S rRNA, ACT, TUA, TUB, eIF, H4, UBQ, rps4, and GAPDH, using 34 RNA samples of C. ehrenbergii cultured in various environments (e.g. exposure to heat shock, UV, metals, and non-metallic chemicals). Each housekeeping gene tested displayed different ranges of C T values for each experimental condition. The gene stability was determined using the descriptive statistic software geNorm, which showed that ACT, H4, and TUA were the most suitable reference genes for all the conditions tested. In addition, at least three genes were required for proper normalization. With these references, we assessed the expression level of the heat shock protein 70 (HSP70) gene in C. ehrenbergii cells exposed to thermal and toxic contaminant stress and found that it was significantly up-regulated by these stressors. This study provides potential reference genes for gene expression studies on C. ehrenbergii with qRT-PCR.

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“…Results were given as the mean of three biological replicates (three technical replicates for each qRT-PCR reaction). RPL19 and GAPDH were set as reference genes for qRT-PCR analysis (Liu et al 2012).…”

Section: Rna Isolation and The Expression Profiles Of Rna Helicase Genesmentioning

confidence: 99%

Transcriptome-wide analysis of DEAD-box RNA helicase gene family in an Antarctic psychrophilic alga Chlamydomonas sp. ICE-L

Liu

Huang

2015

Extremophiles

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DEAD-box RNA helicase family proteins have been identified in almost all living organisms. Some of them play a crucial role in adaptation to environmental changes and stress response, especially in the low-temperature acclimation in different kinds of organisms. Compared with the full swing study in plants and bacteria, the characters and functions of DEAD-box family proteins had not been surveyed in algae. To identify genes critical for freezing acclimation in algae, we screened DEAD-box RNA helicase genes from the transcriptome sequences of a psychrophilic microalga Chlamydomonas sp. ICE-L which was isolated from Antarctic sea ice. Totally 39 DEAD-box RNA helicase genes had been identified. Most of the DEAD-box RNA helicase have 1:1 homologous relationships in Chlamydomonas reinhardtii and Chlamydomonas sp. ICE-L with several exceptions. The homologous proteins in ICE-L to the helicases critical for cold or freezing tolerance in Arabidopsis thaliana had been identified based on phylogenetic comparison studies. The response of these helicase genes is not always identical in the Chlamydomonas sp. ICE-L and Arabidopsis under the same low-temperature treatment. The expression of several DEAD-box RNA helicase genes including CiRH5, CiRH25, CiRH28, and CiRH55 were significantly up-regulated under freezing treatment of ICE-L and their function in freezing acclimation of ICE-L deserved further investigation.

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Validation of housekeeping genes for gene expression studies in an ice alga Chlamydomonas during freezing acclimation

Cited by 46 publications

References 34 publications

Validation of reference genes for cryopreservation studies with the gorgonian coral endosymbiont Symbiodinium

Validation of reference genes for cryopreservation studies with the gorgonian coral endosymbiont Symbiodinium

Evaluation and selection of reference genes for ecotoxicogenomic study of the green alga Closterium ehrenbergii using quantitative real-time PCR

Transcriptome-wide analysis of DEAD-box RNA helicase gene family in an Antarctic psychrophilic alga Chlamydomonas sp. ICE-L

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