Validation of seven housekeeping genes as reference ones for qRT-PCR normalization in Dendrobium catenatum

Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-PCR measurements strongly depends on the normalization with appropriate endogenous reference genes. In this study a set of candidate reference genes for the use in gene expression studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or cadmium was identified, evaluated and validated. Seven candidate genes were selected from cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has its own most stably expressed set of reference genes, regardless of the metal treatments, in casu metal exposures. Metal treatments had only a minor impact on the expression of the candidate reference genes. The validated reference genes outperform the in fungal research commonly used single, arbitrary chosen (“housekeeping”) genes in terms of reliability, and have the potential to be suitable reference genes when studying the effect of other environmental factors. A relatively high number of reference genes is required to correct for intraspecific variability when studying natural populations.

Download Full-text

Selection of housekeeping genes for qRT-PCR analysis in potato tubers under cold stress

Molecular Breeding ◽

10.1007/s11032-012-9766-z ◽

2012 ◽

Vol 31 (1) ◽

pp. 39-45 ◽

Cited By ~ 26

Author(s):

Raquel Lopez-Pardo ◽

Jose Ignacio Ruiz de Galarreta ◽

Enrique Ritter

Keyword(s):

Cold Stress ◽

Housekeeping Genes ◽

Pcr Analysis ◽

Potato Tubers ◽

Qrt Pcr ◽

Selection Of

Download Full-text

Identification of reference genes for qRT-PCR studies of gene expression in giant cells and syncytia induced in Arabidopsis thaliana by Meloidogyne incognita and Heterodera schachtii

Nematology ◽

10.1163/156854107781352034 ◽

2007 ◽

Vol 9 (3) ◽

pp. 317-323 ◽

Cited By ~ 23

Author(s):

Florian Grundler ◽

Julia Hofmann

Keyword(s):

Gene Expression ◽

Arabidopsis Thaliana ◽

Reference Genes ◽

18S Rrna ◽

Housekeeping Genes ◽

Nematode Infection ◽

Heterodera Schachtii ◽

Parasitic Nematodes ◽

Root Knot Nematode ◽

Qrt Pcr

AbstractSedentary plant-parasitic nematodes, such as cyst and root-knot nematodes, induce feeding structures in the host root that undergo extensive changes in the gene expression. This phenomenon has previously been studied by gene chip analysis and qRT-PCR. Housekeeping genes are often used routinely as internal references for relative qRT-PCR analyses. However, due to the strong influence of nematode infection on host cell metabolism and physiology, expression of housekeeping genes may be altered considerably, thus limiting reliability of qRT-PCR analyses. Therefore, in the present work we tested UBQ10, ACT2, EF1a, UBP22 and 18S rRNA as potential candidates for relative qRT-PCR studies of gene expression in nematode infection sites in roots of Arabidopsis thaliana. Among the tested candidates only UBP22 and 18S rRNA were stably expressed and, therefore, are reliable reference genes for studying cyst and root-knot nematode infections.

Download Full-text

Characterisation and validation of housekeeping genes for qRT-PCR expression analysis in Pterophyllum scalare

Aquaculture International ◽

10.1007/s10499-021-00754-x ◽

2021 ◽

Author(s):

Ngairangbam Sushila ◽

Basanta Kumar Das ◽

K. Pani Prasad ◽

Gayatri Tripathi

Keyword(s):

Expression Analysis ◽

Housekeeping Genes ◽

Qrt Pcr ◽

Pterophyllum Scalare

Download Full-text

Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii

PeerJ ◽

10.7717/peerj.6834 ◽

2019 ◽

Vol 7 ◽

pp. e6834

Author(s):

Teizhu Yang ◽

Bingning Gu ◽

Guolyu Xu ◽

Yanmei Shi ◽

Heding Shen ◽

...

Keyword(s):

Gene Expression ◽

Osmotic Pressure ◽

Reference Gene ◽

Salinity Stress ◽

Reference Genes ◽

Target Gene ◽

Housekeeping Genes ◽

Target Gene Expression ◽

Qrt Pcr ◽

Suitable Reference

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.

Download Full-text

Reference genes for RT-qPCR normalisation in different tissues, developmental stages and stress conditions of Hypericum perforatum

10.1101/499186 ◽

2018 ◽

Author(s):

Zhezhi Wang ◽

Wen Zhou ◽

Lei Yang ◽

Yan Sun ◽

Qian Zhang ◽

...

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Reference Genes ◽

Hypericum Perforatum ◽

Developmental Stages ◽

Housekeeping Genes ◽

Potential Candidate ◽

Qrt Pcr ◽

Wide Range ◽

Different Tissues

Hypericum perforatum is a widely known medicinal herb used mostly as a remedy for depression because of its abundant secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is very necessary for the interpretation of qRT-PCR data. In this study, we investigated the expression of fourteen candidate genes, including nine housekeeping genes and five potential candidate genes. Additionally, the HpHYP1 gene, belonging to the PR-10 family associated with stress control, was used for validation of the candidate reference genes. Three programs were applied to evaluate the gene expression stability across four different plant tissues, three developmental stages and a set of abiotic stress and hormonal treatments. The candidate genes showed a wide range of Ct values in all samples, indicating that they are differentially expressed. Integrating all of the algorithms and evaluations, ACT2 and TUB-β were the most stable combination overall and for different developmental stages samples. Moreover, ACT2 and EF1-α were considered to be the two most applicable reference genes for different tissues and for stress samples. Majority of the conventional housekeeping genes exhibited better than the potential reference genes. The obtained results will contribute to improving credibility of standardization and quantification of transcription levels in future expression research of H. perforatum.

Download Full-text

Selection of optimal reference genes for quantitative RT-PCR transcript abundance analysis in white clover (Trifolium repens L.)

Functional Plant Biology ◽

10.1071/fp17304 ◽

2018 ◽

Vol 45 (7) ◽

pp. 737 ◽

Cited By ~ 7

Author(s):

Rafael Narancio ◽

Ulrik John ◽

John Mason ◽

German Spangenberg

Keyword(s):

White Clover ◽

Trifolium Repens ◽

Reference Genes ◽

Mrna Level ◽

Housekeeping Genes ◽

Transcript Abundance ◽

Mrna Levels ◽

Qrt Pcr ◽

Trifolium Repens L ◽

Suitable Reference

Quantitative reverse transcription PCR (qRT-PCR) is a widely used method for transcript abundance analyses in plants. Relative quantification by qRT-PCR requires the use of a stably expressed reference gene. There are many ‘housekeeping’ genes reported in different plant species that are used as reference genes. However, it is important that the steady-state mRNA levels of these housekeeping genes are confirmed across different conditions and tissues in each species studied. Prior to this study, no comprehensive work had been performed in identifying optimal reference genes in white clover (Trifolium repens L.). To identify suitable reference genes in white clover, we analysed the transcript abundance stability of seven candidate genes in two organs (leaves and stolons) across two treatments (water-limited and well-watered). ΔCt, NormFinder and ANOVA tests were carried out to evaluate the mRNA level stability of candidate reference genes. According to the ΔCt results, the genes with the most stable mRNA levels were EF1α and ACT11. When stability among groups was evaluated by NormFinder, UBQ was the most stable across all organs and treatments. By multiple criteria, EF1α, followed by ACT11 and UBQ, was the most stably-expressed gene across organs and treatments, and each of these are recommended as reference genes for transcript abundance studies in white clover.

Download Full-text

Reference Gene Validation in the Brain Regions of Young Rats after Pentylenetetrazole-Induced Seizures

Biomedicines ◽

10.3390/biomedicines8080239 ◽

2020 ◽

Vol 8 (8) ◽

pp. 239

Author(s):

Alexander P. Schwarz ◽

Anna A. Kovalenko ◽

Daria A. Malygina ◽

Tatiana Y. Postnikova ◽

Olga E. Zubareva ◽

...

Keyword(s):

Entorhinal Cortex ◽

Reference Genes ◽

Dorsal Hippocampus ◽

Quantitative Polymerase Chain Reaction ◽

Temporal Cortex ◽

Housekeeping Genes ◽

Ventral Hippocampus ◽

Brain Regions ◽

Qrt Pcr ◽

The Right

Reverse transcription followed by quantitative polymerase chain reaction (qRT-PCR) is a powerful and commonly used tool for gene expression analysis. It requires the right choice of stably expressed reference genes for accurate normalization. In this work, we aimed to select the optimal reference genes for qRT-PCR normalization within different brain areas during the first week following pentylenetetrazole-induced seizures in immature (P20–22) Wistar rats. We have tested the expression stability of a panel of nine housekeeping genes: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on geometric averaging of ranks obtained by four common algorithms (geNorm, NormFinder, BestKeeper, Comparative Delta-Ct), we found that the stability of tested reference genes varied significantly between different brain regions. The expression of the tested panel of genes was very stable within the medial prefrontal and temporal cortex, and the dorsal hippocampus. However, within the ventral hippocampus, the entorhinal cortex and amygdala expression levels of most of the tested genes were not steady. The data revealed that in the pentylenetetrazole-induced seizure model in juvenile rats, Pgk1, Ppia, and B2m expression are the most stable within the medial prefrontal cortex; Ppia, Rpl13a, and Sdha within the temporal cortex; Pgk1, Ppia, and Rpl13a within the entorhinal cortex; Gapdh, Ppia, and Pgk1 within the dorsal hippocampus; Rpl13a, Sdha, and Ppia within the ventral hippocampus; and Sdha, Pgk1, and Ppia within the amygdala. Our data indicate the need for a differential selection of reference genes across brain regions, including the dorsal and ventral hippocampus.

Download Full-text

Gene expression studies in different genotypes of an ectomycorrhizal fungus require a high number of reliable reference genes

10.7287/peerj.preprints.2125 ◽

2016 ◽

Author(s):

Joske Ruytinx ◽

Tony Remans ◽

Jan V Colpaert

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Intraspecific Variability ◽

Standard Technique ◽

Housekeeping Genes ◽

Ectomycorrhizal Fungus ◽

Suillus Luteus ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-PCR measurements strongly depends on the normalization with appropriate endogenous reference genes. In this study a set of candidate reference genes for the use in gene expression studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or cadmium was identified, evaluated and validated. Seven candidate genes were selected from cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has its own most stably expressed set of reference genes, regardless of the metal treatments, in casu metal exposures. Metal treatments had only a minor impact on the expression of the candidate reference genes. The validated reference genes outperform the in fungal research commonly used single, arbitrary chosen (“housekeeping”) genes in terms of reliability, and have the potential to be suitable reference genes when studying the effect of other environmental factors. A relatively high number of reference genes is required to correct for intraspecific variability when studying natural populations.

Download Full-text